Medicinal uses of phenylaikanols and derivatives

ABSTRACT

A compound of formula (I), a pharmaceutically acceptable derivative thereof, wherein Ph is a phenyl radical R 1  is H, OH, OC 1-4 alkyl, NO 2 ; R 2  is OH, OC 1-4 alkyl, OC═OC 1-4 alkyl or OC═OPh where the Ph can be optionally substituted by halogen, C 1-3  alkyl or NO2; R 1  and R 2  along with the two carbon atoms of the phenyl ring to which they are attached can combine to form a 5 or 6 membered heterocyclic ring comprising 1 or 2 heteroatoms selected from O, S or N; R 3  is an optionally substituted hydroycarby radical; R 4  is H, CH 3 , OH or ═O; when R 4  is ═O, then the carbon to which R 4  is attached is not bonded to H; W is C(═O)—CH 2 , CH═CH—, CH 2 CO, CH(OH)—CH 2 , C(CH 3 )(OH)CH 2 , CH 2 CH(OH), CH 2 C(CH 3 )OH, CO, CHOH, C(CH 3 )(OH), CH 2 , CH 2 CH 2 ; X is —CH—OH, C(CH 3 )OH, CH 2 , CH(CH 3 ) or —C═O; Y is —CH—OH, C(CH 3 )OH, CH 2 , CH(CH 3 ) or —C═O; provided that one of W, X or Y has an OH group.

This application is a 371 of PCT/AU98/00870 filed Oct. 20, 1998.

TECHNICAL FIELD

The present invention relates to the use of phenylalkanols (gingerolanalogues) in the treatment or prophylaxis of diseases by the inhibitionof platelet aggregation. The present invention further relates to theuse of phenylalkanols (gingerol analogues) in the treatment orprophylaxis of pain by action on sensory nerves and/or throughanti-inflammatory action.

BACKGROUND ART

Agents directly or indirectly controlling calcium are potentially usefulfor the treatment of congestive heart failure, hypertension, pain,diabetes and cancer (Vincenzi, 1981) or may have cardioprotective orneuroprotective properties. Other agents of interest are those known toaffect calcium channel mediated Ca²⁺ uptake into cells, such as thetherapeutic 1,4-dihydropyridine drug nifedipine and verapamil (Triggle,1984). They are useful antianginal drugs as well as antihypertensives.Agents that have anti-inflammatory properties and antiplateletproperties are potentially useful for the treatment of inflammation,pain, stroke and ischaemic diseases.

The gingerols are a series of natural homologues isolated from ginger,Zingiber officinale. Gingerols are classified according to their alkylchain length eg. [6]-gingerol, [8]-gingerol (Deniff et al, 1981). Apatent is published (Takeda et al, 1992) on the preparation of racemicgingerols (eg. [6]-gingerol) and their dehydrated derivatives (eg.[6]-shogaol) and their use as antipyretic and analgesic agents (nodata). Another patent is published (Tanaka et al, 1987) on a shogaolderivative where the carbonyl group of the side-chain is reduced tohydroxy group and its use in the treatment of thrombosis and pain.

Agents that inhibit platelet aggregation may be used for the treatmentof cardiovascular diseases and stroke. Platelets play an essential rolein blood clotting at sites of wound injury, but unwanted activation ofplatelets in the circulation can give rise to thrombus formation, and isimplicated in the onset of stroke, myocardial infarction, and otherdiseases. Therapeutic modalities aimed at secondary prevention of strokeand ischaemic diseases include vascular surgery, anticoagulant andplatelet aggregation inhibition. Among these, the platelet aggregationinhibition appears to be the most promising because in fast-flowingvessels thrombi are composed mainly of platelets with little fibrin.Recent clinical trials have indicated that antiplatelet therapy protectsa wide range of patients at high risk of occlusive vascular disease(Antiplatelet Trialists' Collaboration, 1994). Medium dose aspirin isthe most widely used antiplatelet regimen, and no other regimen appearedsignificantly more effective at preventing myocardial infarction andstroke. However, gastrointestinal tract upset, particularly pepticulcer, is a common problem associated with the use of aspirin (Roderick,1993). In addition, complications in some disease conditions such asdiabetes and asthma are of major concern in the use of aspirin. A newsafe antiplatelet therapy is therefore required.

There is a need for safe and effective agents for the treatment of painand inflammation, particularly arthritis. The use of analgesics such asnon-steroidal anti-inflammatory agents (NSAIDS), paracetamol andmorphine still remain a primary therapy for such conditions. Each ofthese agents, however, has limitations. Aspirin and newer non-steroidalanti-inflammatory agents can cause gastrointestinal discomfort andeventually the development of peptic ulcer. Paracetamol may produceliver and kidney toxicity with chronic use. Morphine, though effective,can be addictive and exhibit tolerance. Recently, a topical analgesichas been developed from capsaicin for control of pain(anti-nociception). Capsaicin has also been used extensively forresearch in neurosciences, where it has benefit in the modulation ofsensory nerve activity (nerves which transmit sensations of pain-causingstimuli from the periphery to the brain). Capsaicin has also yieldedimportant knowledge about pain pathways. However, capsaicin is anirritant and cannot be administered systemically because of itspotential to cause neuro-inflammation. Its use as a topical agent isalso limited for several reasons: it causes mild to moderate burningsensation, erythema and stinging after application; severe irritation tosensitive organs such as eyes; it cannot be used on broken or irritatedskin; excessive inhalation of aerosolised dried cream may causecoughing, which is the most commonly reported systemic side-effectassociated with the use of capsaicin preparations. Higher doses mayproduce neurotoxic effects through mechanisms not completely understood.Development of more effective anti-nociceptive agents is imperative.

Stroke and ischaemic diseases that afflict millions of peopleworld-wide, are among the most common maladies affecting people inindustrialised countries. Current efforts directed at reducing themorbidity and mortality of these disease conditions are aimed at bothrelief and preventative therapies. The platelet aggregation inhibitionappears to be the most promising modality aimed at prevention of strokeand ischaemic diseases because in fast-flowing vessels thrombi arecomposed mainly of platelets with little fibrin.

There is great need to develop more effective drugs with novel action.Substances that are the subject of the present invention are typicallysubstances that exert useful medicinal actions through mechanisms wherecalcium is either directly or indirectly involved. For example,hypertension including stroke are common disorders with extremely highmortality rate. Their incidence is steadily increasing despite asubstantial haemostasis improvement by a number of therapeutic regiments(Salmo, 1995).

DISCLOSURE OF THE INVENTION

In one aspect, the present invention provides a compound of formula (I),a pharmaceutically acceptable derivative thereof:

where

R₁ is H, OH, OC₁₋₄alkyl, NO₂

R₂ is OH, OC₁₋₄alkyl, OC═OC₁₋₄alkyl or OC═OPh where the Ph can beoptionally substituted by halogen, C₁₋₃ alkyl or NO₂;

R₁ and R₂ along with the two carbon atoms of the phenyl ring to whichthey are attached can combine to form a 5 or 6 membered heterocyclicring comprising 1 or 2 heteroatoms selected from O, S or N;

R₃ is C₂₋₁₂alkyl, C₂₋₁₂alkenyl or C₂₋₁₂alkynyl each optionallysubstituted by one or more substituents selected from —OR, ═O, nitro,halogen, —NRR′, —COOR or —CONRR′ where R and R′ are H or C₁₋₄alkyl;

R₃ may be a linking group of a bis compound where R₃ is C₂₋₁₂alkylene,C₂₋₁₂alkenylene or C₂₋₁₂alkynylene each optionally substituted by one ormore substituents selected from —OR, ═O, nitro, halogen, —NRR′, —COOR or—CONRR′ where R and R′ are H or C₁₋₄alkyl;

R₄ is H, CH₃, OH or ═O; when R₄ is ═O, then the carbon to which R₄ isattached is not bonded to H;

W is C(═O)—CH₂, CH═CH—, CH₂CO, CH(OH)—CH₂, C(CH₃)(OH)CH₂, CH₂CH(OH),CH₂C(CH₃)OH, CO, CHOH, C(CH₃)(OH), CH₂, CH₂CH₂;

X is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;

Y is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;

provided that one of W, X or Y has an OH group and provided that when

(1) R₁ is OC₁₋₄alkyl, R₂ is OH or OAcyl, W=CH₂CH₂ and X=C═O, R₃ is C₂₋₁₂alkyl, R₄ is H, then Y is not CHOH (gingerols) (Mustafa et al, 1993);

(2) R₁ is OCH₃, R₂ is OH, W is CH₂CH₂, R₃ is C₅ or C₇ alkyl, R₄ is H andX=CHOH then Y is not CHOH (gingerdiol) (Mustafa et al, 1993);

(3) R₁ is OCH₃, R₂ is OH, W is CH═CH, R₃ is C₂₋₁₂ alkyl, R₄ is H and Xis C═O, then Y is not CHOH (dehydrogingerols);

(4) R₁ is OCH₃, R₂ is OH, W=CH₂CH₂, X is CHOH, R₄ is H and R₃ is C₅alkyl then Y is not CH₂ (reduced paradol) (Young-Joon et al, 1992);

(5) R₁ is OCH₃, R₂ is OH, W=CH₂CH₂, X is C═O, R₄ is H then Y is notC(OH)CH₃ (Sawamura et al).

(6) R₁ is OC₁₋₄ alkyl, R₂ is OH or OAcyl, W=CH═CH and X=C═O, R₃ is C₂₋₈alkyl, R₄ is H, then Y is not CHOH ([4]-[10]-dehydrogingerols) (Denniffet al, 1981);

(7) R₁=R₂ is OH, W=CH₂CH₂ and X=C═O, R₃ is C_(4,6) alkyl, R₄ is H, thenY is not CHOH ([6]- and [8]-norgingerols) (Terumo Corporation, 1992;Meiji, 1989);

8) R₁=R₂ is OH, W=CH═CH and X=C═O, R₃ is C₆ alkyl, R₄ is H, then Y isnot CHOH ([8]-nordehydrogingerols) (Terumo Corporation, 1992);

(9) R₁ is OC₁₋₄ alkyl, R₂ is OH or OAcyl, W=CH₂CH₂ and X=C═O, R₃ isC_(2,4,6) alkyl, R₄ is H, then Y is not C═O ([4]-, [6]- and[8]-gingerdiones) (Denniff et al, 1981); (Terumo Corporation, 1992);

(10) R₁ is OC₁₋₄ alkyl, R₂ is OH or OAcyl, W=CH═CH and X=C═O, R₃ isC_(2,4,6) alkyl, R₄ is H, then Y is not C═O ([4]-, [6]- and[8]-dehydrogingerdione) (Denniff et al, 1981); (Terumo Corporation,1992);

(11) R₁=R₂ is OH, W=CH₂CH₂ and X=C═O, R₃ is C₆ alkyl, R₄ is H, then Y isnot C═O ([8]-norgingerdione) (Terumo Corporation, 1992-EP516082);

(12) R₁=R₂ is OH, W=CH═CH and X=C═O, R₃ is C₆ alkyl, R₄ is H, then Y isnot C═O ([8]-nordehydrogingerdione) (Terumo Corporation, 1992);

(13) R₁=R₂ is OH, W=CH₂CH₂ and X=C═O, R₃ is C₂₋₁₂ alkyl, R₄ is H, then Yis not CHOH (norgingerols) (Terumo Corporation, 1992; Meiji, 1989;Merrell Dow Pharmaceuticals, 1992-EP516082);

(14) R₁ is OC₁₋₄ alkyl or OH, R₂ is OH, W is CH₂CH₂, R₃ is C₂₋₁₂ alkyl,R₄ is H and X is CHOH, then Y is not CHOH (gingerdiols ornorgingerdiols) (Merrell Dow Pharmaceuticals-EP516082).

In a second aspect, the present invention provides the use of a compoundof formula (I):

where

R₁ is H, OH, OC₁₋₄alkyl, NO₂

R₂ is OH, OC₁₋₄alkyl, OC═OC₁₋₄alkyl or OC═OPh where the Ph can beoptionally substituted by halogen, C₁₋₃ alkyl or NO₂;

R₁ and R₂ along with the two carbon atoms of the phenyl ring to whichthey are attached can combine to form a 5 or 6 membered heterocyclicring comprising 1 or 2 heteroatoms selected from O, S or N;

is R₃ is C₂₋₁₂alkyl, C₂₋₁₂alkenyl or C₂₋₁₂alkynyl each optionallysubstituted by one or more substituents selected from —OR, ═O, nitro,halogen, —NRR′, —COOR or —CONRR′ where R and R′ are H or C₁₋₄alkyl;

R₃ may be a linking group of a bis compound where R₃ is C₂₋₁₂alkylene,C₂₋₁₂alkenylene or C₂₋₁₂alkynylene each optionally substituted by one ormore substituents selected from —OR, ═O, nitro, halogen, —NRR′, —COOR or—CONRR′ where R and R′ are H or C₁₋₄alkyl;

R₄ is H, CH₃, OH or ═O; when R₄ is ═O, then the carbon to which R₄ isattached is not bonded to H;

W is C(═O)—CH₂, CH═CH—, CH₂CO, CH(OH)—CH₂, C(CH₃)(OH)CH₂, CH₂CH(OH),CH₂C(CH₃)OH, CO, CHOH, C(CH₃)(OH), CH₂, CH₂CH₂;

X is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;

Y is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;

provided that one of W, X or Y has an OH group

a pharmaceutically acceptable derivative thereof in the treatment orprophylaxis of diseases by the inhibition of platelet aggregation.

In a third aspect, the present invention provides the use of a compoundof formula (I):

where

R₁ is H, OH, OC₁₋₄alkyl, NO₂

R₂ is OH, OC₁₋₄alkyl, OC═OC₁₋₄alkyl or OC═OPh where the Ph can beoptionally substituted by halogen, C₁₋₃ alkyl or NO₂;

R₁ and R₂ along with the two carbon atoms of the phenyl ring to whichthey are attached can combine to form a 5 or 6 membered heterocyclicring comprising 1 or 2 heteroatoms selected from O, S or N;

R₃ is C₂₋₁₂alkyl, C₂₋₁₂alkenyl or C₂₋₁₂alkynyl each optionallysubstituted by one or more substituents selected from —OR, ═O, nitro,halogen, —NRR′, —COOR or —CONRR′ where R and R′ are H or C₁₋₄alkyl;

R₃ may be a linking group of a bis compound where R₃ is C₂₋₁₂alkylene,C₂₋₁₂alkenylene or C₂₋₁₂alkynylene each optionally substituted by one ormore substituents selected from —OR, ═O, nitro, halogen, —NRR′, —COOR or—CONRR′ where R and R′ are H or C₁₋₄alkyl;

R₄ is H, CH₃, OH or ═O; when R₄ is ═O, then the carbon to which R₄ isattached is not bonded to H;

W is C(═O)—CH₂, CH═CH—, CH₂CO, CH(OH)—CH₂, C(CH₃)(OH)CH₂, CH₂CH(OH),CH₂C(CH₃)OH, CO, CHOH, C(CH₃) (OH), CH₂, CH₂CH₂;

X is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;

Y is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;

provided that one of W, X or Y has an OH group

a pharmaceutically acceptable derivative thereof in the manufacture of amedicament for the treatment or prophylaxis of diseases by theinhibition of platelet aggregation.

In a fourth aspect, the present invention provides a pharmaceuticalformulation comprising a compound of formula (I):

where

R₁ H, OH, OC₁₋₄alkyl, NO₂

R₂ is OH, OC₁₋₄alkyl, OC═OC₁₋₄alkyl or OC═OPh where the Ph can beoptionally substituted by halogen, C₁₋₃ alkyl or NO₂;

R₁ and R₂ along with the two carbon atoms of the phenyl ring to whichthey are attached can combine to form a 5 or 6 membered heterocyclicring comprising 1 or 2 heteroatoms selected from O, S or N;

R₁ is C₂₋₁₂alkyl, C₂₋₁₂alkenyl or C₂₋₁₂alkynyl each optionallysubstituted by one or more substituents selected from —OR, ═O, nitro,halogen, —NRR′, —COOR or —CONRR′ where R and R′ are H or C₁₋₄alkyl;

R₃ may be a linking group of a bis compound where R₃ is C₂₋₁₂alkylene,C₂₋₁₂alkenylene or C₂₋₁₂alkynylene each optionally substituted by one ormore substituents selected from —OR, ═O, nitro, halogen, —NRR′, —COOR or—CONRR′ where R and R′ are H or C₁₋₄alkyl;

R₄ is H, CH₃, OH or ═O; when R₄ is ═O, then the carbon to which R₄ isattached is not bonded to H;

W is C(═O)—CH₂, CH═CH—, CH₂CO, CH(OH)—CH₂, C(CH₃)(OH)CH₂, CH₂CH(OH),CH₂C(CH₃)OH, CO, CHOH, C(CH₃)(OH), CH₂, CH₂CH₂;

X is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;

Y is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;

provided that one of W, X or Y has an OH group

a pharmaceutically acceptable derivative thereof in a pharmaceuticallyacceptable carrier.

In a fifth aspect, the present invention provides novel compounds asfollows

1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol

1-(4-hydroxy-3-methoxyphenyl)dodecan-5-ol

3-methyl-1-(4-hydroxy-3-methoxyphenyl)undecan-3-ol

3-methyl-1-(4-hydroxy-3-methoxyphenyl)tridecan-3-ol

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-5-one

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-1-one

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-1-one

1-hydroxy-1-(4-hydroxy-3-methoxyphenyl)undecan-2-one

2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)undecan-1-one

5-hydroxy-1-(2-hydroxy-3-methoxyphenyl)dodecan-3-one ([8]-orthogingerol)

5-hydroxy-1-(4-hydroxyphenyl)decan-3-one

5-hydroxy-1-(4-hydroxyphenyl)dodecan-3-one

5-hydroxy-1-(4-hydroxyphenyl)dodecan-1-ene-3-one

5-hydroxy-1-(3,4-methylenedioxyphenyl) dodecan-3-one

5,12-dihydroxy-1,16-bis(4-hydroxy-3-methoxyphenyl)hexadecane-3,14-dione(a bis compound)

1-(4-hydroxy-3-methoxyphenyl)dodecane-1,4-diene-3-one.

2-hydroxy-1-(3,4-dimethoxyphenyl)dodecan-3-one

2-hydroxy-1-(3,4-dimethoxyphenyl)undecan-4-one

1-(3,4-dimethoxyphenyl)dodecan-2-ol

All alkyl, alkenyl, alkynyl, alkylene, alkenylene and alkynylene carbonchains can be straight or branched chain.

Halogen includes bromo, chloro, fluoro or iodo.

Pharmaceutically acceptable derivatives include acid addition salts.

In a further aspect, the present invention provides the use of acompound of formula (I) according to the second aspect of the presentinvention in the treatment or prophylaxis of pain by action on sensorynerves and/or through anti-inflammatory action and/or through neurokinininhibitory action.

Preferably, the use of a compound of formula (I) in the treatment orprophylaxis of pain by action on sensory nerves is as an analgesic.

In another aspect, the present invention provides the use of a compoundof formula (I) according to the second aspect of the present inventionin the treatment or prophylaxis of cardiovascular disease.

In yet another aspect, the present invention provides a process forpreparing the following compounds

which comprises treating ginger extract with heat 10 and/or acid andthen followed by treatment with a microorganism or enzyme.

MODES FOR CARRYING OUT THE INVENTION

Starting materials for preparing compounds of formula (I) arecommercially available or are prepared according to literatureprocedures.

The following description provides methods of preparing compounds offormula (I).

(1) when W is —CH═CH—, X is C═O, Y is —CHOH— and R₃ is alkyl, alkenyl oralkynyl

(i) treating the appropriate benzaldehyde with acetone

(ii) protecting any hydroxy groups

(iii) treating the resulting compound with an appropriate aliphaticaldehyde in the presence of LDA as follows

 and deprotecting as necessary;

(2) when W is —CH₂CH₂—, X is C═O, Y is —CHOH— and R₃ is alkyl or whereR₃ is a linking group of a bis compound and R₃ is alkylene

reducing the product obtained in (1) above;

 or when R₃ is alkyl, alkenyl or alkynyl or where R₃ is a linking groupof a bis compound and R₃ is alkenylene or alkynylene

reducing the intermediate ketone compound from (1) above beforecondensation with the appropriate aldehyde as follows:

(3) when W is CH═CH, X is CHOH and Y is C═O starting with theappropriate cinnamaldehyde and reacting to protect any hydroxy groups ifnecessary and then treating with appropriate ketone in LDA as follows

 and deprotecting as necessary;

(4) when W is CH₂CH₂, X is CHOH, Y is C═O and R₃ is alkyl

reducing the product of (3) above;

 or when R₃ is alkyl, alkenyl or alkynyl starting with the appropriatecinnamaldehyde and reducing as for (2) above before condensation withthe appropriate ketone or alternatively oxidising the appropriatealcohol followed by condensation with the appropriate ketone as follows

5) when W is C(═O)—CH₂, X is CHOH and Y is CH₂ starting with theappropriate acetophenone compound and protecting any hydroxy groups ifnecessary and treating with the appropriate aldehyde compound as follows

 and deprotecting as necessary

(6) when W is CH₂, X is CO and Y is CHOH

(7) when W is CHOHCH₂, X is CO and Y is CH₂

(8) when W is CH₂, X is CHOH and Y is CO

(9) when W is CO and COCH₂, X is CH₂ and Y is CHOH

(10) when W is CH₂CO, X is CH₂ and Y is CHOH

(11) when W is CHOH and CHOHCH₂, X is CH₂ and Y is C═O

(12)when W is CH₂CHOH, X is CH₂ and Y is CO

 Substances with the α-hydroxyketone group may be prepared by thefollowing general procedure (Organic Syntheses 3, 562)

(13) when W is CO, X is CHOH and Y═CH₂

(14) when W is CH₂CO, X is CHOH and Y is CH₂

(15) when W is CHOH, X is CO and Y is CH₂

(16) when W is CH₂CHOH, X is CO and Y is CH₂

(17) when W is CH₂, CH₂CH2 or other having an OH group, Y is CHOH or CH2and X is CH₂ or CHOH but one of W, X or Y has an OH group

 or alternatively when W does not have an OH group and X is CHOH or CH,and Y is CHOH or CH₂ but one of X or Y is CHOH

(18) when W is CHOH or CH₂CHOH, X is CH₂ and Y is CH₂

(19) when W is CH₂CH₂, X is CHOH and Y is CH₂, then in the formula in(18) n=2;

(20) when W is CH₂ or CH₂CH₂, and X is CH₂ and Y is CHOH then in theformula in (18) n=3 or 4.

For Grignard or similar condensation, the aldehyde can be replaced withthe methyl ketone to give the methyl branched product.

In the preparation of α-hydroxyketones the cyanohydrin may be preparedfrom a methylketone.

Reduction steps are typically carried out with hydrogen using a suitablecatalyst such as Pd/C or with NaBH₄ or NaCNBH₃.

Oxidation steps are typically carried out using pyridiniumchlorochromate.

Protecting groups are typically tetrahydropyran (THP) or acyls.

Asymmetric Synthesis of Gingerol Analogues

Asymmetric synthesis of the gingerol and analogues can be achievedeither by organic chemistry or enzyme-catalysed reaction. The mostattractive features of using enzymes in asymmetric synthesis are thatenzymes are inherently chiral and have ability to catalyse reactionswith high selectivity leading to the synthesis of single stereoisomers.The asymmetric transformation of a prochiral ketone group (shown inreaction scheme below) into highly optical pure R- or S-isomer can beachieved either by organic synthesis or enzyme-catalysed reaction. Inorganic synthesis, R- or S-isomer can be exclusively formed by catalytichydrogenation catalysed by an enantiomeric form of transition metalcomplexes. In the present invention ruthenium (R)-(+)-BINAP andruthenium (S)-(−)-BINAP complexes will be employed as chiral catalysts(Noyori and Takaya, 1990). Alternatively, R- and S-isomer of thegingerol analogues can be formed by enzyme-catalysed reduction. In thiscase a separate enzyme system can be employed to produce optically pureenantiomers. Two enzyme systems that can be used in this reaction areAspergillus niger and Thermoanaerobium brockii alcohol dehydrogenase(Belan, et al, 1987), (Faber, 1995; Turner, 1996) as shown in thereaction scheme below.

Asymmetric Synthesis of Capsaicin-like Analogues

Asymmetric synthesis of capsaicin-like analogues can be achieved by thecondensation of an aldehyde with an α-ketoacid. The reaction is known asacyloin condensation which is effectively catalysed by an enzymaticsystem, pyruvate decarboxylase (Crout, et al. 1991), (Faber, 1995;Turner, 1996). The advantage of using this enzyme is a remarkabletolerance by the enzyme system with respect to a range of structures ofthe aldehyde. More significantly from a synthetic point of view, theα-hydroxyketone compounds can be converted chemically or enzymaticallyinto the corresponding dione or chiral diol compounds.

R can be: alkyl, alkenyl, alkynyl, phenylalkyl, phenylalkenyl,phenylalkynyl, etc. R may have methyl branches and/or substitutions.

Transformation of Ginger Preparations, Gingerols, Gingerol Analogues andRelated Substances Using Enzymes or Micro-organisms to ProduceTherapeutically Useful Products

Ginger extract contains numerous phenolic substances. Many of thesesubstances are present as optically pure isomers, for example, [6]- and[8]-gingerols, the most abundant pungent components present in freshginger extract have been identified as the (−)-(S)-isomers. Gingerolspossess a β-hydroxy keto functional group which makes them vulnerable todegradation by dehydration to form shogaols. This degradation perhaps isthe major cause of loss in potential therapeutic effect of gingerols andthe variations and changes in therapeutic effects of gingerpreparations. The degradation of gingerol, however, results in theformation of the biologically active component, shogaol, which hasdifferent pharmacological activities to gingerol. This biologicallyactive component may be enzymatically transformed into variouscomponents in ginger which are yet to be fully determined. Theenzyme-catalysed conversion of [6]-shogaol in vitro using a supernatantfraction isolated from rat liver is reported to result in the formationof [6]-paradol, [6]-dehydro- and [6]-dihydroparadol. An homologue of[6]-dihydroparadol was chemically synthesised in our laboratory, namely,1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol [3.93] which was found to havea range of therapeutically useful biological activities. The gingerextract can be treated with yeast or isolated enzyme to formdihydroshogaol, dihydroparadol, and other reduced derivatives including1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol [3.93]. Compounds of formula(I) can also be prepared by treating the ginger extract with heat, acid,enzymes or micro-organisms, or combination or sequence thereof, toproduce products that contain optimal amounts of dihydroshogaol,dihydroparadol (particularly 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-oland homologues) or other substances with similar therapeutic actions.The optimally transformed ginger extract can be used to producetherapeutically useful herbal medicines and pharmaceutical agents.

The process described above enables a more stable, potent and effectiveproduct to be derived from ginger preparations based on thetherapeutically useful actions of gingerols and gingerol like substancesfalling within the scope of the general formula (I). This process isparticularly suited to the production of herbal products.

Transformation of a Ginger Preparation Using Yeast

Yeast is a convenient source of enzymes that has been extensivelyexploited in asymmetric synthesis. Its enzyme-catalysed reactions areregarded as natural processes and usually occur under mild conditionsand with attendant selectivity, such as chemo-, regio- andstereoselectivity, leading to the formation of naturally occurringisomers. Apart from the naturally occurring substances such asgingerols, shogaols, gingerdiols, etc. the following compounds can beproduced from the ginger extract in the presence of yeast:

wherein n=1-10

Note: Both the baker's yeast and isolated enzyme such asThermoanaerobium brockii ADH will produce naturally occurring isomers,typically the (S)-isomers (Belan, et al, 1987).

Preferably the acid is a strong acid such as HCl, H₂SO₄ H₃PO₄ and thelike.

Synthetic Gingerol Analogues as Antiplatelet Agents

Racemic gingerols and about 30 analogues have been prepared by synthesisand their biological activities, particularly on the cardiovascularsystem, have been investigated (Tran, 1997). The gingerol analogue,3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane [3.93], was tested on apharmacological screen with arachidonic acid as substrate in rabbitplatelet-rich plasma and found to have potent antiplatelet aggregationactivity, being three times more potent than the indomethacin referencecompound. However, the analogue had little or no effect on bleeding timein mice. Gingerols and synthetic analogues were tested on platelet richplasma from human blood and found to inhibit platelet aggregation.

Effects of Gingerol Analogues on Sensory Neurons

The increase in intracellular calcium level is also known to beimportant for capsaicin-induced desensitisation in rat cultured dorsalroot ganglion neurons (Cholewinski et al, 1993) and is believed, invivo, to give rise to an analgesic effect. Capsaicin is known to excitea subset of sensory neurons by opening non-selective cation channels(Bevan and Szolcsanyi, 1990) which preferentially allows Ca²⁺ ion entryleading to pronounced desensitisation. A synthetic gingerol analogue wasfound to antagonise the effect of capsaicin, and vice versa, in ratmesenteric artery bed. It is proposed that gingerol and its analoguesact on a so called “gingerol receptor” which previously was undefined,or on the capsaicin receptor or a subclass of capsaicin receptor.Pharmacological comparison has been made between capsaicin and[6]-shogaol, a dehydration product of [6]-gingerol (Suekawa et al,1986). The neuropharmacological properties of gingerol and syntheticanalogues may be investigated by specific measurement of Ca²⁺ within thecytosol, nucleus and mitochondria or of Ca²⁺ currents. Specificmeasurements of the action of gingerol and synthetic analogues in thesensory neurons have led to the discovery of new pharmacological agentswith less pungent effect and little or no neuro-inflammatory effectcompared with capsaicin which may be developed as a superior analgesicagent. These agents may be useful for the treatment of pain orconditions such as arthritis.

Anti-inflammatory Action of Gingerol Analogues

Gingerol analogues exert anti-inflammatory action through inhibition oflipoxygenase and cyclooxygenase enzymes and through their antioxidantproperties (Musuda et al, 1995). The gingerol analogues may be used totreat inflammatory conditions such as arthritis and may also be used toprotect against stroke (Munsiff et al, 1992).

Neurokinin-1 Receptor Activity of Gincerol Analogue

A gingerol analogue, 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol [3.93],exhibited relatively potent inhibition of neurokinin-1 receptor (NK-1)mediated by substance P. Gingerol analogues may exert theirantinociceptive and anti-inflammatory activities through this mechanismand, therefore, may be useful in the treatment of pain and inflammatoryconditions such as migraine headache and internal pain.

Known Substances

5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one ([6]-gingerol)

5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-3-one ([8]-gingerol)

5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-1-ene-3-one([8]-dehydrogingerol)

1-(4-hydroxy-3-methoxyphenyl)dodecan-4-ene-3-one ([8]-shogaol)

1-(4-hydroxy-3-methoxyphenyl)dodecan-3-one ([8]-paradol)

1-(4-hydroxy-3-methoxyphenyl)dodecane-3,5-diol ([8]-gingerdiol)

5-hydroxy-1-(3-hydroxy-4-methoxyphenyl)dodecan-3-one ([8]-isogingerol)

New Chemical Entities

1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol

1-(4-hydroxy-3-methoxyphenyl)dodecan-5-ol

3-methyl-1-(4-hydroxy-3-methoxyphenyl)undecan-3-ol

3-methyl-1-(4-hydroxy-3-methoxyphenyl)tridecan-3-ol

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-5-one

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-1-one

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-1-one

1-hydroxy-1-(4-hydroxy-3-methoxyphenyl)undecan-2-one

2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)undecan-1-one

5-hydroxy-1-(2-hydroxy-3-methoxyphenyl)dodecan-3-one ([8]-orthogingerol)

5-hydroxy-1-(4-hydroxyphenyl)decan-3-one

5-hydroxy-1-(4-hydroxyphenyl)dodecan-3-one

5-hydroxy-1-(4-hydroxyphenyl)dodecan-1-ene-3-one

5-hydroxy-1-(3,4-methylenedioxyphenyl)dodecan-3-one

5,12-dihydroxy-1,16-bis(4-hydroxy-3-methoxyphenyl)hexadecane-3,14-dione

1-(4-hydroxy-3-methoxyphenyl)dodecane-1,4-diene-3-one

2-hydroxy-1-(3,4-dimethoxyphenyl)dodecan-3-one

2-hydroxy-1-(3,4-dimethoxyphenyl)undecan-4-one

1-(3,4-dimethoxyphenyl)dodecan-2-ol

A number of structural analogues of phenolic hydroxyketones calledgingerols (listed above) were prepared by synthesis.1-(4-Hydroxy-3-methoxyphenyl)dodecan-3-ol [3.93] emerged as one of themost interesting substances. It was initially identified as the mostpotent inotropic agent in the guinea pig atrium and it was thought thatits inotropic activity was a result of enhancing of SR Ca²⁺-ATPase sinceit exhibited relatively potent SR Ca²⁺-ATPase activation. It was foundhowever that the positive inotropic effect in the series could bedissociated from the enhancement of SR Ca²⁺ pump stimulation (Tran,1997). This is in contrast to the previous reports (Kobayashi et al,1988) which had led to the conclusion that the positive inotropic(increased force of contraction) effect of [8]-gingerol on the guineapig atrium was associated with the stimulation of Ca²⁺ uptake into thesarcoplasmic reticulum (SR) of cells via the SR Ca²⁺ pump, therebyallowing greater Ca²⁺ release (and hence force of contraction) onstimulation of the myocardium. Further work with1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol showed that the positiveinotropic effect of the compound was produced through actions on sensorynerves innervating the release of neuropeptides and possibly histamine.Most importantly, the inotropic effect of the compound was blocked bypretreatment of the atrium with capsaicin, and pretreatment of theatrium with the compound caused a loss of capsaicin inotropic effect. Aputative capsaicin receptor antagonist, capsazepine (10 μM) (Bevan etal, 1992) was found to block the inotropic response to1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol completely. These results areconsistent with a mechanism whereby both capsaicin and1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol [3.93] cause an increase inrate and force of guinea pig atria by releasing calcitonin gene relatedprotein (cGRP) (and possibly other neuropeptides) from sensory nerves,which in turn acts directly on the atria and/or indirectly by therelease of histamine (Imamura et al, 1996). However, it is not clearwhether capsaicin and the compound exert their effects via the samereceptor, by different subsets of capsaicin receptors or by a differentpathway yet to be described. A relationship between capsaicin and1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol is supported by a comparisonof the structures of the two compounds, which are vanilloids showingsignificant similarities (but also differences). The inotropic activityof 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol was shown to beenantiospecific as one enantiomer exhibited 100 fold more potent actionthan the other in increasing the force of contraction in guinea pigatria.

The proposed mechanism of neuropeptide release by1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol, similar to that of capsaicin,was supported by studies in blood vessels. The compound was shown to bevery potent in relaxing vasopressin-contracted rat mesenteric smallarteries (200-300 μm diameter) at 10⁻⁸ to 10⁻⁶ M. This effect wasantagonised by capsaicin pretreatment (3×10⁻⁷ M). Interestingly, thiseffect on relaxation of mesenteric artery could not be explained by cGRPrelease, using a selective cGRP antagonist, even though cGRP is known torelax this arterial bed. The potency (EC₅₀) of the compound forrelaxation of the mesenteric artery was 100-fold greater than for theinotropic effect in guinea pig atrium. This contrasts with capsaicinwhich has similar activity in Guinea pig atria (force and rate) andrelaxing rat mesenteric artery.

In contrast to inotropic activity of1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol which is enantiospecific, bothenantiomers showed similar potency in relaxation of rat mesentericvascular bed. Preliminary investigation on vasodilation property ofother gingerol analogues such as3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-5-one,5-hydroxy-1-(3,4-methylenedioxyphenyl)dodecan-3-one and [8]-paradol, allshowed potency in relaxing rat mesenteric artery.

In summary, these results suggest that1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol [3.93] acts like capsaicin torelease one or more vasorelaxant substances from sensory nerves.

The gingerol analogue, 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane[3.93], was tested on a pharmacological screen with arachidonic acid assubstrate in rabbit platelet-rich plasma and found to have potentantiplatelet aggregation activity, three time more potent than theindomethacin reference compound. However, the analogue had little or noeffect on bleeding time in mice. Antiplatelet activity of the compoundwas shown to be specific for arachidonic acid as the substance did notaffect platelet function either via adenosine diphosphate or thromboxaneA mechanisms. It is therefore thought that the compound has interferedwith arachidonic acid metabolism, probably by inhibiting cyclo-oxygenaseenzyme. Gingerols and synthetic analogues were tested on platelet richplasma from human blood and found to inhibit platelet aggregationinitiated by arachlidonic acid.

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane [3.93] also showedinhibition of 5-lipoxygenase from rat basophilic leukemia cells (RBL-1)with arachidonic acid as substrate.

The gingerol analogue, 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane[3.93],was tested on guinea pig submaxillary membrane for neurokinin-1(NK-1) antagonist activity with tritium labelled [³H]Substance P andfound to have relatively potent inhibition of the binding of Substance Pto NK-1 receptor.

Acute toxicity of the gingerol analogue,3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane [3.93] was evaluated onmice for 3 days and found, for intraperitoneal administration, to causea slight decrease in spontaneous activity, response to touch and limbtone in mice. However, no toxicity was shown towards mice for dose bythe oral route. The compound showed low toxicity towards brine shrimp.Brine shrimp toxicity for gingerols and gingerol analogues tested rangedfrom low to moderate.

Discussion of Results

We have reviewed evidence and preliminary work showing that gingerolsand capsaicin, which are members of the vanilloid chemical family, mayact by similar mechanisms in producing positive inotropic effects onguinea pig heart and vasorelaxation in rat mesenteric artery. However,it is unknown whether they act on the same receptors, on relatedreceptor subtypes or on different but linked receptors. There isconsiderable information on the nature of capsaicin receptors, butnothing is known about the site of action of gingerols. In both groupsof compounds the 4-hydroxy-3-methoxy (vanilloid) substitution isessential for biological activity. We showed that the side chain couldbe modified by changing the relationship between the keto and hydroxysubstituent.

The vasorelaxant effect of gingerol analogue3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane [3.93] appears to beunrelated to release of cGRP, the major vasorelaxant peptide with othervasorelaxants studied. Operation of a novel vasorelaxant pathway issuggested by these results. The established action of capsaicin inanti-nociception, probably related to depletion of the neurotransmitterSubstance P from sensory nerves, and hence tolerance of the relay ofpain sensation via afferent nerve pathways to the central nervoussystem, indicates a role for capsaicin derivatives (includingpotentially 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane [3.93] andother derivatives) as candidate analgesic agents and for inhibition ofneurogenic inflammation (Wrigglesworth et al, 1996). In addition, thegingerol analogues, as shown by3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane [3.93], may exert theirantinociceptive activity by inhibition of Substance P from binding toNK-1 receptor.

A number of novel substances (gingerol analogues) have been found thatare much more chemically stable than the gingerols which are relativelyunstable under both chemical (Mustafa et al., 1993) and biological(Young-Joon, 1992, 1994) conditions, forming inactive substances. Theβ-hydroxycarbonyl function of the gingerols is vulnerable to oxidationor dehydration (Mustafa et al, 1993) to form inactive products. Thegingerols are particularly prone to rapid dehydration under acidicconditions (Mustafa et al, 1993) such that even the pure substance isdifficult to store for long periods. Simple oral dosing of the gingerolsfor medicinal action would not be possible due to the acidic environmentof the stomach and upper intestinal tract. Chemical and biologicalinstability is also likely to be a serious problem for intravenousdoses.

Other useful bioactivities and properties have been reported forgingerols and related substances, for example, antipyretic,antihepatotoxic (Hikino et al, 1985) and antischistosomal activities(Young-Joon, 1992, 1994; Suekawa et al, 1984), antiulcer (Yamahara etal, 1992; Yoshikawa et al, 1992) and antioxidant activities (Aeschbachet al, 1994). The action of gingerols and chemically related substancesin suppression of spontaneous calcium spikes and contraction in isolatedportal veins of mice has also been reported (Kimura et al, 1988).

In work carried out in our laboratories, guinea pig atria organ bathtests did not give the predicted results, suggesting that the proposedmode of action of this class of compounds (Kobayashi et al., 1988) needsto be reinvestigated. A gingerol analogue3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decanone [3.92], an isomer of[6]-gingerol), has potent stimulatory activity towards dog heart SRCa²⁺-ATPase (200% at 3 μM and 95% at 25 μM for the gingerol analogue[3.92]), whereas preliminary results from the guinea pig atria organbath studies showed negative inotropic activity and negativechronotropic activity. In later studies positive inotropic activity wasobserved as a 50% increase on driven guinea pig left atria at 10 μM.

Positive inotropic and chronotropic activity were observed for othergingerol analogues [see Table 1].3-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)decanone [3.92] showed 50%increase on guinea pig driven left atria at 10 μM and3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane [3.93] showed 50%increase at 1 μM followed by arrhythmia. Neither blocked ATP orα₁-receptors in vas deferens. No effect was observed on nervestimulation in atria.

3-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)decanone [3.92] at up to 10 μMshowed only a small effect on the rate of rise of Na⁺-dependent actionpotential, amplitude of action potential, and duration of actionpotential (at 50% or 90% recovery).

Cardiotonic Substances of Interest

Of particular interest are gingerol analogues of novel structure showingcardiotonic activity (Table 1) i.e.

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-5-one([8]-inversegingerol) [3.90],

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecanone [3.91],

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decanone [3.92],

1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol [3.93].

Of potential interest is3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecanone [3.91]. This substanceis a homologue of the cardiotonic3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decanone [3.92].

ATPase activities are a subject of this investigation. Filling of the SRstores by stimulation of SR Ca²⁺,-ATPase may be of benefit in enhancingcardiac contractility whereas simultaneous stimulation of the PMCa²⁺-ATPase may aid in relaxation during diastole. Compounds of thisclass may be of considerable interest.

Our research includes mechanisms which directly control the level ofintracellular calcium which is important for excitation-contractioncoupling. There have been reports that [8]-gingerol, isolated fromginger, specifically activates sarcoplasmic reticulum (SR) Ca²⁺-ATPaseat low concentrations but inhibits the enzyme at high concentrations(Kobiyashi et al, 1987). [8]-Gingerol was also found to exhibitrelatively potent cardiotonicity towards guinea pig atria.

This observation was confirmed by our laboratory for both [6]- and[8]-gingerol. Although the mechanism of the cardiotonic action has beenreported to be the result of activation of SR Ca²⁺-ATPase evidence forthis is circumstantial or indirect, therefore the mechanism of action isuncertain. Evidence that the SR Ca²⁺-ATPase may not be directly involvedin the cardiotonic action comes from the observation of a very rapiddose-dependent response 15-20 seconds after addition of the gingerol tothe guinea pig atria organ bath. A very rapid onset of action isunlikely to be due to activation of SR Ca²⁺-ATPase which is located deepwithin the cell. Other evidence comes from studies of gingerol analogueswhere cardiotonic action does not correlate with activation of SRCa²⁺-ATPase and in some cases cardiotonic gingerol analogues showedinhibitory activity towards SR Ca²⁺-ATPase.

The gingerol analogues may be useful for the treatment of heart failurethrough increase in strength of contraction of the heart. Also ofparticular interest with regard to cardiotonic activity of the gingerolanalogues (and the gingerols) is the increase in relaxation of theguinea pig atria observed in the diastolic phase (observed as a decreasein the baseline tension after addition of the compound in the assay).This activity may be of use in the treatment of diastolic heart failure.

EXPERIMENTAL

I. Synthesis of Gingerols and Their Derivatives

1. Preparation of Dehydrogingerone

To a solution of vanillin (5 g) in acetone (20 mL) was added 10% sodiumhydroxide solution (20 mL). The reaction mixture was stirred at roomtemperature for 4 days (Normura, 1917). After acidification the productwas extracted with EtOAc twice and washed with water. Evaporation ofsolvent left a dark brown liquid which on crystallisation fromEtOAc-petroleum afforded the title compound (85%).

2. Preparation of Dehydrogingerone-THP (2)

A mixture of dehydrogingerone (5 g) and pyridinium p-toluene sulfonate(PPTS ) (0.1 g) in dichloromethane (20 mL) was stirred at roomtemperature for 24 hrs (Miyashita et al., 1977). After removal ofsolvent, the crude product was subjected to gradient chromatography togive a colourless solid (92%) which was sufficiently pure for the nextreaction.

3. Preparation of 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one([6]-gingerol)

To a stirred solution of LDA (0.15 moles, prepared by treatingdiisopropylamine with 2.5 M butyllithium in hexane), under N₂, in THF (4mL) and HMPA (1 mL) at −78° C. was added dropwise a solution of 2 (0.1moles) in THF (2 mL). After stirring for 20 mins, an appropriatealdehyde (0.12 moles) in THF (1 mL) was added dropwise. The reactionmixture was stirred at −78° C. for overnight, extracted with Et₂O twice,washed with diluted HCl, then with water. Evaporation of solvent left ayellowish liquid which was subjected to gradient chromatography to givethe dehydrogingerol-THP. The product subsequently underwenthydrogenation at room conditions with hydrogen and Pd—C, anddeprotection of the THP ether, using PPTS in ethanol, to afford a lightyellow liquid which was purified by gradient chromatography to give acolourless liquid. Yield 60%.

¹H-NMR: δ6.81 (1H, d, J=8 Hz), 6.67 (2H, m), 4.02 (1H, m), 3.86 (3H, s,OCH₃), 2.77 (4H, m), 2.52 (2H, m), 1.28 (8H, m), 0.88 (3H, t, b).¹³C-NMR: δ14.03, 22.59, 25.13, 29.27, 31.72, 36.41, 45.43, 49.34, 55.86,67.66, 110.97, 114.38, 120.71, 132.62, 143.95, 146.43.

CI-MS {M+1}⁺ 295 (15), {M+1−H₂O}⁺ 277 (100), {C₁₀H₁₁O₃}⁺ 179 (30),{C₈H₉O₂}⁺ 137 (35).

A similar procedure was applied to the synthesis of other gingerolderivatives.

5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-1-ene-3-one([8]-dehydrogingerol): mp. ¹-NNR: δ7.56 (1H, d, J=15 Hz), 7.11 (1H, dd,J=8, 2 Hz), 7.06 (1H, d, J=2 Hz), 6.94 (1H, d, J=8 Hz), 6.58 (1H, d,J=15 Hz), 4.02 (1H, m), 3.87 (3H, s, OCH₃), 2.78 (2H, m), 1.51 (2H, m),1.29 (10H, m), 0.89 (3H, t, b).

5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-3-one ([8]-gingerol):Liquid.

¹H-NMR: δ6.81 (1H, d, J=8 Hz), 6.67 (2H, m), 4.02 (1H, m), 3.86 (3H, s,OCH₃), 2.77 (4H, m), 2.52 (2H, m), 1.28 (12H, m), 0.88 (3H, t, b).¹³C-NMR: δ14.03, 22.64, 25.45, 29.27, 29.24, 29.28, 29.49, 31.79, 36.45,45.43, 49.34, 55.86, 67.66, 110.97, 114.38, 120.71, 132.62, 143.95,146.43.

CI-MS: {M+1}⁺ 323 (15), {M+1−H₂O}⁺ 305 (100), {C₁₀H₁₁O₃}⁺ 179 (20),{C₈H₉O₂}⁺ 137 (20).

5-hydroxy-1-(2-hydroxy-3-methoxyphenyl)dodecan-3-one: ([8]-o-gingerolprepared using o-vanillin instead of vanillin as starting material).

Liquid. ¹H-NMR: δ6.75 (3H, m), 4.02 (1H, m), 3.86 (3H, s, OCH₃), 2.77(4H, m), 2.52 (2H, m), 1.28 (12H, m), 0.88 (3H, t, b). ¹³C-NMR: δ14.03,22.70, 25.51, 29.29, 29.56, 31.85, 36.45, 43.41, 49.08, 49.99, 56.02,67.66, 108.92, 119.55, 122.34, 126.57, 143.56, 146.54.

CI-MS: {M+1−H₂O}⁺ 305 (30), {C₁₀H₉O₃}⁺ 177 (100).

EI-MS: {M} 322 (5), {M−H₂O} 304 (20), {C₁₂H₁₃O₃} 205 (30), {C₁₂H₁₈O₂}194 (20), {C₁₀H₁₀O₃} 178 (25), {C₈H₉O₂} 137 (60), {C₅H₅O} 81 (25),{C₄H₉} 69 (60), {C₄H_(9}) 57 (40), {C₃H₅} 41 (100).

HRMS: C₁₉H₃₀O₄ Calculated 322.214, Found 322.214.

5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-3-one: ([8]-isogingerolprepared using isovanillin instead of vanillin as starting material).

Liquid. ¹H-NMR: δ6.75 (2H, m), 6.64 (1H, dd, J=8, 2 Hz), 4.02 (1H, m),3.86 (3H, s, OCH₃), 2.77 (4H, m), 2.52 (2H, m), 1.26 (12H, m), 0.88 (3H,t, b). ¹³C-NMR: δ14.15, 22.70, 25.51, 28.99, 29.29, 29.55, 31.85, 36.48,45.21, 49.31, 56.04, 67.68, 110.73, 114.44, 119.68, 134.01, 145.03,145.61.

CI-MS: {M+1}⁺ 323 (10), {M+1−H₂O}⁺ 305 (100), {C₁₀H₁₁O₃}⁺ 179 (40),{C₈H₉O₂}⁺ 137 (18).

5-hydroxy-1-(4-hydroxyphenyl)decan-3-one: ([6]-demethoxygingerolprepared using 4-hydroxybenzaldehyde instead of vanillin as startingmaterial).

mp 43-45° C. ¹H-NMR. δ7.02 (2H, d, J=8 Hz), 6.73 (2H, d, J=8 Hz), 4.03(1H, m), 2.77 (4H, m), 2.52 (2H, m), 1.28 (8H, m), 0.88 (3H, t, b).¹³C-NMR: δ14.07, 22.63, 25.13, 28.73, 31.72, 36.41, 45.37, 49.29, 67.81,115.41, 129.42 (2C), 132.70 (2C), 154.11.

CI-MS: {M+1−H₂O}⁺ 247 (30), {C₁₁H₁₃O₂}⁺ 177 (100).

EI-MS: {M} 264 (10), {M−H₂O} 246 (20), {C₁₁H₁₁O₂} 175 (80), {C₈H₈O} 120(40), {C₇H₇O} 107 (100), {C₄H₇} 55 (100), {C₃H₅} 41 (55).

HRMS: C₁₆H₂₄O₃ Calculated 264.173, Found 264.172.

5-hydroxy-1-(4-hydroxyphenyl)dodecan-3-one: ([8]-demethoxygingerol,preparation similar to that of [6]-demethoxygingerol).

mp 81-82° C. ¹H-NMR: δ6.81 (2H, d, J=8 Hz), 6.67 (2H, d, J=8 Hz), 4.02(1H, m), 3.86 (3H, s, OCH₃), 2.77 (4H, m), 2.52 (2H, m), 1.28 (12H, m),0.88 (3H, t, b). ¹³C-NMR: δ14.03, 22.64, 25.45, 29.27, 29.24, 29.28,29.49, 31.79, 36.45, 45.43, 49.34, 55.86, 67.66, 110.97, 114.38, 120.71,132.62, 143.95, 146.43.

CI-MS: {M+1−H₂O}⁺ 275 (60), {C₉H₉O₂}⁺ 149 (25), {C₇H₇O}⁺ 107 (100).

EI-MS: {M} 292 (18), {M−H₂O} 274 (18), {C₁₁H₁₁O₂} 175 (65), {C₉H₉O₂} 149(20), {C₈H₈O} 120 (60), {C₇H₇O} 107 (100), {C₅H₉} 69 (30), {C₄H₇} 55(60), {C₃H₇} 43 (90).

HRMS: C₁₈H₂₈O₃ Calculated 292.204, Found 292.205.

5-hydroxy-1-(3,4-methylenedioxyphenyl)dodecan-3-one: [prepared usingpiperonal instead of vanillin as starting material]

mp 48-50° C. ¹H-NMR: δ6.72 (1H, d, J=8 Hz), 6.66 (1H, d, J=2 Hz), 6.62(1H, dd, J=8, 2 Hz), 5.92 (2H, s, OCH₂O), 4.03 (1H, m), 2.75 (4H, m),2.53 (2H, m), 1.27 (12H, m), 0.88 (3H, t, b). ¹³C-NMR: δ14.14, 22.69,25.50, 29.28, 29.31, 29.54, 31.84, 36.51, 45.35, 49.36, 67.68, 100.89,108.32, 108.80, 121.08, 134.55, 145.92, 147.70.

CI-MS: {M+1−H₂O}⁺ 303 (50), {C₁₁H₁₃O₃}⁺ 193 (25), {C₈H₇O₂}⁺ 135 (100),{C₈H₁₅}⁺ 111 (50).

EI-MS: {M} 320 (40), {M−H₂O} 302 (60), {C₁₂H₁₁O₃} 203 (70), {C₉H₈O₂} 148(40), {C₈H₇O₂} 135 (100), {C₅H₉} 69 (30), {C₄H₆} 54 (50), {C₃H₆} 42(45).

5,12-dihydroxy-1,16-bis(4-hydroxy-3-methoxyphenyl)-hexa-decane-3,14-dione:

[prepared using 1,8-octandial instead of aliphatic aldehyde as startingmaterial]

mp 65-68° C. ¹H-NMR: (CD₃COCD₃) δ6.82 (2H, d, J=2 Hz), 6.71 (2H, d, J=8Hz), 6.65 (2H, dd, J=8, 2 Hz), 4.01 (2H, m), 3.81 (6H, S, OCH₃), 2.77(8H, m), 2.52 (4H, m), 1.30 (12H, m). ¹³C-NMR: (CD₃COCD₃) δ26.18 (2C),38.1 (2C), 38.14 (2C), 45.86 (2C), 50.89 (2C), 50.94 (2C), 56.14 (2C),68.15 (1C), 68.28 (1C), 112.72 (2C), 115.51 (2C), 115.6 (2C), 121.39(2C), 133.61 (2C).

CI-MS: {M+1}³⁰ 531 (100), {M+1−H₂O)⁺ 513 (70), {M+1−2H₂O}⁺ 495 (15),{C₁₉H₂₇O₄}⁺ 319 (50), {C₁₀H₁₁O₃}⁺ 179 (10), {C₈H₉O₂}⁺ 137 (80).

EI-MS: C₁₂H₁₃O₃} 205 (10), {C₁₁H₁₄O₃} 194 (15), {C₉H₁₀O₂} 150 (15),{C₈H₉O₂} 137 (100), {C₄H₇} 55 (25), {C₃H₇} 43 (80).

II. Synthesis of 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-5-one

1. Preparation of 4-benzyloxy-3-methoxycinnamaldehyde

4-Hydroxy-3-methoxycinnamaldehyde (0.6 g) was added to the mixture ofbenzyl chloride (1 mL), K₂CO₃ (1 g), and NaI (1 g) in acetone (20 mL).The resulting mixture was stirred at room temperature for 24 hrs. Thesolid were removed by filtration and washed with acetone. Afterevaporation of solvent, the crude product was purified by gradientchromatography to give a yellowish solid. Yield 85%.

2. Preparation of 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-5-one

To a stirred solution of LDA (0.15 moles, prepared by treatingdiisopropylamine with 2.5 M butyllithium in hexane), under N₂, in THF (4mL) and HMPA (1 mL) at −78° C., was added dropwise a solution of2-nonanone (0.1 moles) in THF (1 mL). After stirring for 20 mins,4-benzyloxy-3-methoxycinnamaldehyde (0.11 moles) in THF (2 mL) was addeddropwise. The reaction mixture was stirred at −78° C. overnight, thenquenched with dilute HCl, extracted with Et₂O twice. Evaporation ofsolvent left a yellowish liquid which was subjected to gradientchromatography to give a yellow solid. The product subsequentlyunderwent hydrogenation at room temperature and atmospheric pressurewith hydrogen and Pd—C for 2 hrs to afford the title compound which wasthen purified by gradient chromatography to give a colourless solid.Yield 60%. mp 42-43° C. ¹H-NMR: δ6.84 (1H, d, J=8 Hz), 6.72 (1H, d, J=2Hz), 6.70 (1H, dd, J=8, 2 Hz), 4.05 (1H, m), 3.88 (3H, s, OCH₃), 2.60(4H, m), 2.41 (2H, t, J=6 Hz), 1.60 (4H, m), 1.27 (8H, m), 0.88 (3H, t,b). ¹³C-NMR: δ14.11, 22.64, 23.67, 29.08, 29.16, 31.50, 31.69, 38.41,43.72, 48.93, 55.92, 66.92, 111.10, 114.26, 120.95, 133.83, 143.73,146.41.

CI-MS: {M+1}⁺ 323 (100), {M+1−H₂O}⁺ 305 (60), {C₁₀H₁₁O_(2l }) ⁺ 163(20), {C₈H₉O₂}⁺ 137 (40), {C₈H₁₅O}⁺ 127 (30).

EI-MS: {M} 322 (80), {M−H₂O} 304 (50), {C₁₂H₁₃O₃} 205 (10), {C₁₁H₁₃O₂}177 (18), {C₁₀H₁₁O₂} 163 (25), {C₉H₁₀O₂} 150 (20), {C₈H₉O₂} 137 (100),{C₈H₁₅O} 127 (25).

HRMS: C₁₉H₃₀O₄ Calculated 322.214, Found 322.215.

III. Synthesis of 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decanone

To a stirred solution of LDA (0.15 moles, prepared by treatingdiisopropylamine with 2.5 M butyllithium in hexane), under N₂, in THF at−78° C was added dropwise a solution of acetovanillone-THP (0.1 moles),which was prepared as described for dehydrogingerone-THP, in THF (4 mL).After stirring for 20 mins, octanal (0.12 moles) in THF (2 mL) was addeddropwise. The reaction mixture was stirred at −78° C. overnight, thenquenched with dilute HCl and extracted with ether twice. Evaporation ofsolvent left a yellowish liquid which was subjected to gradientchromatography to give the product. This was subsequently deprotectedusing PPTS in ethanol, to afford a light yellow liquid which was againsubjected to gradient chromatography to give the title compound as acolourless solid. Yield 70-80%. mp 77-78° C. ¹H-NMR: δ7.53 (2H, m), 6.94(1H, d, J=8 Hz), 4.19 (1H, m), 3.96 (3H, s, OCH₃), 3.10 (2H, m), 1.30(12H, m), 0.88 (3H, t, b). ¹³C-NMR: δ14.15, 22.71, 25.66, 29.33, 29.64,31.88, 36.62, 44.41, 56.13, 68.07, 109.63, 113.94, 123.74, 129.84,146.73, 150.83, 199.64.

EI-MS: {M} 294 (10), {M−H₂O} 276 (10), {C₁₀H₁₁O₄} 195 (15), {C₉H₁₀O₃}166 (40), {C₈H₇O₃} 151 (100), {C₇H₇O₂} 123 (10).

HRMS: C₁₇H₂₆O₄ Calculated 294.183, Found 294.185.

A similar procedure to the above was applied to synthesise the followingcompound.

3-hydroxy-1-(4-hydroxy-1-methoxyphenyl)dodecanone: mp 74-76° C. ¹H-NMR:δ7.53 (2H, m), 6.94 (1H, d, J=8 Hz), 4.19 (1H, m), 3.96 (3H, s, OCH₃),3.10 (2H, m), 1.30 (16H, m), 0.88 (3H, t, b). ¹³C-NMR: δ14.15, 22.71,25.66, 29.33, 29.64, 29.67, 29.74, 31.85, 36.62, 44.41, 56.13, 68.07,109.63, 113.94, 123.74, 129.84, 146.73, 150.84.

CI-MS: {M+1}⁺ 323 (100), {M+1−H₂O}⁺ 305 (25), {C₈H₇O₃}⁺ 151 (20).

MS-EI: {M} 322 (20), {M−H₂O} 304 (15), {M −47} 279 (15), {C₁₀H₁₁O₄} 195(25), {C₉H₁₀O₃} 166 (40), {C₈H₇O₃} 151 (100), {C₇H₇O₂} 123 (10).

HRMS: C₁₉H₃₀O₄ Calculated 322.214, Found 322.213.

IV. Synthesis of 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol

To a solution of 8-dehydrogingerol (0.1 g) in dichloromethane (20 mL)was added anhydrous p-toluenesulfonic acid (0.05 g ). The mixture wasstirred at room temperature overnight. Evaporation of solvent left adark brown liquid which subsequently was hydrogenated with H₂/Pd—C, thenreduced with sodium borohydride in ethanol to produce the title compoundin quantitative yield.

mp 66—68° C. ¹H-NMR: δ6.84 (1H, d, J=8 Hz), 6.72 (1H, d, J=2 Hz), 6.69(1H, dd, J=8, 2 Hz), 3.89 (3H, s, OCH₃), 3.63 (1H, m), 2.70 (2H, m),1.74 (2H, m), 1.47 (2H, m), 1.27 (14H, m), 0.89 (3H, t, b). ¹³C-NMR:δ14.17, 22.73, 25.68, 29.37, 29.61, 29.67, 29,74, 31.85, 31.94, 37.70,39.43, 55.91, 71.49, 111.00, 114.26, 120.91, 134.17, 143.68, 146.41.

CI-MS: {M+1}⁺ 309 (10), {M+1−H₂O}⁺ 291 (100), {C₈H₉O₂}⁺ 137 (55).

EI-MS: {M} 308 (65), {M−H₂O} 290 (25), {C₉H₁₀O₂} 150 (25), {C₈H₁₀O₂} 138(100), {C₇H₈O₂} 124 (12), {C₄H₇} 43 (55).

HRMS: C₁₉H₃₂O₃ Calculated 308.235, Found 308.234.

1-(4-hydroxy-3-methoxyphenyl)dodecane-1,4-diene-3-one: (isolated as anintermediate product). Liquid. ¹H-NMR: δ7.59 (1H, d, J=15 Hz), 7.14 (1H,dd, J=8, 2 Hz), 7.08 (1H, d, J=2 Hz), 7.02 (1H, m), 6.93 (1H, d, J=8Hz), 6.82 (1H, d, J=15 Hz), 6.45 (1H, m), 3.94 (3H, s, OCH₃), 2.29 (2H,m), 1.51 (2H, m), 1.31 (8H, m), 0.88 (3H, t, b). ¹³C-NMR: δ14.14, 22.69,28.27, 29.14, 29.26, 31.8, 32.78, 56.01, 109.74, 114.88, 122.83, 123.34,127.41, 129.08, 143.37, 146.88, 148.08, 148.21, 189.35.

EI-MS: {M} 302 (100), {M−17} 285 (15), {M−31} 271 (10), {C₁₄H₁₅O₃} 231(10), {C₁₃H₁₃O₃} 217 (100), {C₁₂H₁₂O₃} 204 (30), {C₁₂H₉O₂} 185 (20),{C₁₀H₉O₃} 177 (40), {145 (30), {C₈H₉O₂} 137 (60), {117 (20), 89 (20),{49 (15), {C₄H₇} 55 (60), {C₃H₇} 43 (90).

HRMS: C₁₉H₂₆O₃ Calculated 302.188, Found 302.189.

A similar procedure to the above was applied to prepare its homologue.

1-(4-hydroxy-3-methoxyphenyl)dodecane-5-ol: mp 54-55° C. ¹H-NMR: δ6.82(1H, d, J=8 Hz), 6.67 (2H, m), 3.88 (3H, s, OCH₃), 3.60 (1H, m), 2.56(2H, t, J=6 Hz), 1.65 (2H, m), 1.35 (16H, m), 0.88 (3H, t, b). ¹³C-NMR:δ14.16, 22.72, 25.36, 25.71, 29.35, 29.72, 31.89, 31.91, 35.67, 37.35,37.58, 55.90, 71.99, 111.01, 114.20, 120.91, 134.64, 143.59, 146.37.

EI-MS: {M} 308 (100), {M−H₂O} 290 (50), {C₈H₉O₂} 137 (75), }C₅H₉} 69(10), {C₄H₇} 55 (25).

HRMS: C₁₉H₃₂O₃ Calculated 308.235, Found 308.236.

V. Synthesis of 1-(4-hydroxy-3-methoxyphenyl)dodecane-3,5-diol([8]-gingerdiol)

To a solution of [8]-gingerol (0.1 g) in EtOH (15 mL) was added dropwisea solution of NaBH₄ (0.02 g in 1 mL H₂O). The mixture was stirred atroom temperature for 3 hrs. After acidification, EtOAc was added andorganic layer was washed with water twice. Evaporation of solvent left awhite solid which was purified by gradient chromatography to afford thetitle compound as a colourless liquid in quantitative yield.

¹H-NMR: δ6.82 (1H, d, J=8 Hz), 6.71 (1H, d, J=2 Hz), 6.67 (1H, dd, J=8,2 Hz), 3.97 (2H, m), 3.86 (3H, s, OCH₃), 2.65 (2H, m), 1.81 (2H, m),1.69 (14H, m), 0.87 (3H, t, b).

¹³C-NMR: δ14.13, 22.69, 24.99, 29.29, 29.34, 29.55, 30.90, 31.84, 37.22,39.22, 55.91, 71.77, 72.74, 111.20, 114.34, 120.98, 133.73, 143.74,146.47.

CI-MS: {M+1}⁺ 325 (25), {M+1−H₂O}⁺ 307 (45), {M+1−2H₂O}⁺ 289 (100),{C₁₀H₁₁O₂}⁺ 163 (20), {C₈H₉O₂}⁺ 137 (50).

Preparation of 3-methyl-1-(4-hydroxy-3-methoxyphenyl)-undecan-3-ol

To a Grignard solution of octylmagnesiumbromide, prepared from Mg (0.05g) and 1-bromooctane (0.36 g), in THF (5 ml) under N₂ was addedgingerone-THP (0.5 g) in THF (5 ml) which was prepared as described fordehydrogingerone-THP above. The mixture was stirred at room temperatureovernight. The product was extracted with diethyl ether (50 ml), washedwith brine solution and purified by column chromatography to give acolourless liquid (yield 60%).

¹-NMR: δ6.82 (1H, dd, J=8, 2 Hz), 6.7(2H, m), 5.47 (1H, s), 3.88 (3H,s), 2.6 (2H, m), 1.73 (2H, m), 1.5 (2H, m), 1.2-13 (16H, m), 0.88 (3H,t, b). ¹³C-NMR: δ14.17, 22.73, 24.03, 26.93, 29.34, 29.66, 30.05, 30.28,31.94, 42.13, 43.98, 55.91, 72.67, 110.93, 114.23, 120.81, 134.57,143.55, 146.36.

CI-MS: {M+1}⁺ 291, {C₈H₉O₂}⁺ 137; EI-MS: {M} 290.

Similar procedure was used to synthesise3-methyl-1-(4-hydroxy-3-methoxyphenyl)tridecan-3-ol.

¹H-NMR: δ6.82 (1H, dd, J=8, 2 Hz), 6.7(2H, m), 5.49 (1H, s), 3.88 (3H,s), 2.6 (2H, m), 1.73 (2H, m), 1.5 (2H, m), 1.2-13 (20H, m), 0.88 (3H,t, b). ¹³C-NMR: δ14.18, 22.74, 24.04, 26.99, 29.4, 29.68 (3C), 30.06,30.28, 31.96, 42.18, 44.02, 55.91, 72.8, 110.93, 114.3, 120.81, 134.59,143.65, 146.43.

CI-MS: {M+1}⁺ 319, {C₈H₉O₂}⁺137; EI-MS: {M}⁺ 318.

Preparation of 2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)undecan-1-one

1. Preparation of1-(N,N-dimethylamino)-1-(4-hydroxy-3-methoxyphenyl)acetonitrile

The synthesis followed a published method (Hauser et al., 1960).Briefly, to a stirred solution of NaHSO₃ (0.7 g) in water (4 ml) wasadded vanillin-THP (1.4 g), prepared as described fordehydrogingerone-THP above, in MeOH (20 ml), followed by the addition ofanhydrous dimethylamine (0.5 g) in cold MeOH (30 ml). The mixture wascooled prior to the addition of an aqueous solution of NaCN (0.5 g, 2ml). After 24 hrs stirring at room temperature, the mixture wasextracted with Et₂O (50 ml), washed with water (2×20 ml), and evaporatedto give a colourless liquid (yield >90%) which was sufficiently pure forthe next reaction.

2. Preparation of 2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)undecan-1-one

Under a N₂ atmosphere diisopropylamine (0.6 mL) dissolved in dry THF (10mL) was treated with n-butyllithium (2.5 M, 2 mL) and stirred for 30 minat −80° C., followed by the addition of a solution of1-(N,N-dimethylamino)-1-(4-hydroxy-3-methoxyphenyl)acetonitrile (0.8 g),which was prepared as described above, in THF (2 ml). The mixture wasthen stirred at −80° C. for 15 mins and at 0° C. for 2 hr. To thismixture was cooled to −80° C. and then a solution of decyl aldehyde(0.25 g) in THF (2 mL) was added dropwise. After 2 hrs stirring at −80°C., the mixture was extracted with Et₂O (50 ml), washed with brinesolution (20 ml) and evaporated to give a liquid which was purified bycolumn chromatography to afford a colourless liquid (yield 60%).

mp 78-80° C.; ¹H-NMR: δ7.53 (1H, d, J=2 Hz), 7.45 (1H, dd, J=8, 2 Hz),6.96 (1H, d, J=8 Hz), 5.02 (1H, m), 3.97 (3H, s), 3.7 (1H, m), 1.84 (1H,m), 1.5-1.6 (3H, m), 1.23 (12H, m), 0.86 (3H, t, b). ¹³C-NMR: δ14.16,22.71, 24.98, 29.33, 29.44, 29.51, 29.54, 31.91, 36.61, 56.18, 72.67,110.36, 114.11, 123.88, 126.35, 146.91, 151.13.

CI-MS: {M+1}⁺ 309; EI-MS: {M} 308.

Similar procedure was used to synthesise1-hydroxy-1-(4-hydroxy-3-methoxyphenyl)undecan-2-one where1-(N,N-dimethylamino)-1-decylcyanide was formed instead and reacted withvanillin-THP.

mp 51-53° C. ¹H-NMR: δ6.91 (1H, d, J=8 Hz), 6.85 (1H, dd, J=8, 2 Hz),6.72 (1H, d, J=2 Hz), 5.7 (1H, s), 5.0 (1H, d, J=4 Hz), 4.32 (1H, d, J=4Hz), 3.87 (3H, s), 2.33 (2H, m), 1.5 (2H, m), 1.21 (12H, m), 0.86 (3H,t, b). ¹³C-NMR: δ14.14, 22.69, 23.78, 29.04, 29.25 (2C), 29.39, 31.88,37.78, 56, 79.44, 109.06, 114.64, 121.23, 130, 146.1, 147.07.

CI-MS: {M+1}⁺ 309; EI-MS: {M} 308.

Separation of Enantiomers of 1-(4-hydroxy-3-mothoxyphenyl)dodecan-3-ol

1. Preparation of endo-(−)- and(+)-1,4,5,6,7,7-hexachlorobicyclo[2.2.1]hept-5-ene-2-carboxylic acid(HCA). The synthesis followed a published method (Duke and Wells, 1987)in which the diastereomeric esters of HCA were formed using2,3-O-isopropylidene-D(+)-ribono-1,4-lactone and subsequently separatedby repeated fractional crystallisation from hexane/ethyl acetate to givecolourless solids. The diastereomeric esters of HCA was hydrolysed togive endo-(−)- and (+)-HCA, respectively.

2. Preparation of diastereomeric esters of1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol. endo-(+)-HCA (0.34 g) wasrefluxed with SOCl₂ (10 ml) for 1.5 hrs and the excess reagent wasremoved under vacuum. To the residue in THF (5 ml) was added a solutionof 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol (0.1 g) in dry THF (5 ml)and then p-dimethylaminopyridine (0.16 g) in THF (5 ml) was added slowlyto the solution. A colourless solid was formed and the mixture was leftstanding for 3 hrs, then filtered, washed with THF and evaporated togive a colourless liquid. Two diastereomeric esters were separated bycolumn chromatography, then hydrolysed to give, respectively, the twoenantiomers of 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol in quantitativeyield.

Diastereomer-1 of 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol

¹H-NMR: δ6.95 (1H, d, J=8 Hz), 6.78 (2H, m), 4.97 (1H, m), 3.94 (1H, m),3.8 (3H, s), 3.62 (1H, m), 2.5-2.85 (6H, m), 1.89 (2H, m), 1.56 (2H, m),1.25 (14H, m), 0.88 (3H, t, b).

Diastereomer-2 of 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol ¹H-NMR:δ6.96 (1H, d, J=8 Hz), 6.75 (2H, m), 4.95 (1H, m), 3.94 (1H, m), 3.8(3H, s), 3.47 (1H, m), 2.49-2.85 (6H, m), 1.9 (2H, m), 1.56 (2H, m),1.26 (14H, m), 0.88 (3H, t, b).

The exact configuration of each enantiomer has not yet been determined.They are therefore named as enantiomer-1 (less polar) and 2 (more polar)according to the polarity of their diastereomeric esters on normal phasesilica gel chromatography.

enantiomer-1 of 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol:

mp 53-56° C. ¹H-NMR: δ6.82 (1H, dd, J=8,2 Hz), 6.70 (2H, m), 3.87 (3H,s), 3.62 (1H, m), 2.5-2.8 (2H, m), 1.74 (2H, m), 1.46 (2H, m), 1.26(14H, m), 0.87 (3H, t, b). ¹³C-NMR: δ14.16, 22.72, 25.67, 29.36, 29.6,29.66, 29.73, 31.83, 31.94, 37.68, 39.41, 55.91, 71.5, 111.02, 114.28,120.92, 134.17, 143.7, 146.43.

enantiomer-1 of 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol:

mp 53-56° C. ¹H-NMR: δ6.82 (1H, dd, J=8,2 Hz), 6.70 (2H, m), 3.87 (3H,s), 3.62 (1H, m), 2.5-2.8 (2H, m), 1.74 (2H, m), 1.46 (2H, m), 1.26(14H, m), 0.87 (3H, t, b). ¹³C-NMR: δ14.16, 22.72, 25.67, 29.36, 29.6,29.67, 29.73, 31.84, 31.94, 37.7, 39.43, 55.91, 71.5, 111, 114.26,120.92, 134.18, 143.69, 146.42.

Synthesis of 2-hydroxy-1-(3,4-dimethoxyphenyl)dodecan-3-one Preparationof 2-(3,4-dimethoxyphenyl)ethanol

To a solution of 3,4-dimethoxyphenylacetic acid (2 g) in anhydrous THF(40 ml) at 0° C. under N₂ was added dropwise borane-methyl sulfidecomplex (10 M, 1.5 ml). The mixture was stirred at room temperature forfurther 4 hrs. Cold water (5 ml) was added to destroy any excess ofborane followed by the addition of H₂SO₄ (1 M, 50 ml). The mixture wasextracted three time with ethyl acetate (50 ml). The organic layer wasseparated and evaporated off to give a colourless liquid which was thenpurified by column chromatography to afford a colourless solid inquantitative yield.

¹H-NMR: δ6.76-6.82 (3H, m), 3.87 (8H, m), 2.82 (2H, t, J=6 Hz).

Preparation of 3,4-dimethoxyphenyl acetal

To a stirred suspension of pyridinium chlorochromate (2.5 g) in CH₂Cl₂(40 ml) at room temperature was added a solution of2-(3,4-dimethoxyphenyl)ethanol (2 g) in CH₂Cl₂ (10 ml) . The mixture wasstirred at room temperature for further 30 min, then filtered throughflorisil. The solvent was evaporated off to give a liquid which waspurified from column chromatography to afford a colourless liquid. Yield40 %. ¹H-NMR: δ9.73 (1H, t, J=2 Hz), 6.86 (1H, d, J=8 Hz), 6.77 (1H, dd,J=8, 2 Hz), 6.71 (1H, d, J=2 Hz), 3.88 (6H, s), 3.63 (2H, d, J=2 Hz).

Preparation of 2-hydroxy-1-(3,4-dimethoxyphenyl)dodecan-3-one

To a solution of diisopropylamine (0.4 mL) in anhydrous THF (10 mL) at−80° C., under N₂ was added dropwise n-butyllithium (2.5 M, 1.5 mL). Themixture was stirred on ice for 30 min, then cooled to −80° C. prioraddition of the solution 1-(N,N-dimethylamino)-1-decylcyanide (0.36 g),which was prepared as described in the synthesis of2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)undecan-1-one (page 39), in THF(5 ml). The reaction mixture was stirred at −80° C. for 15 mins and at0° C. for further 3 hr. To this mixture cooled at −80° C. was addeddropwise a solution of 3,4-dimethoxyphenylacetal (0.4 g) in THF (5 mL).After 2 hrs stirring at −80° C., the mixture was extracted twice withEt₂O (50 ml), washed with 1 M HCl (50 ml), then water (50 ml). Theorganic layer was evaporated to give a liquid which was purified bycolumn chromatography to afford a colourless liquid (yield 40%).

¹H-NMR: δ6.78 (3H, m), 4.38 (1H, m), 3.86 (3H, s), 3.85 (3H, s), 3.06(1H, m), 2.83 (1H, m), 2.48 (2H, m), 1.58 (2H, m), 1.26 (14H, m), 0.88(3H, t, b). ¹³C-NMR: δ14.15, 22.7, 23.56, 29.27, 29.29, 29.4, 29.43,31.89, 38.66, 39.8, 55.88, 55.91, 77.3, 111.22, 112.53, 121.26, 129.09,148.03, 148.92. CI-MS: {M+1}⁺ 337 (20), {M+1−H₂O}⁺ 319 (70), {C₉H₁₁O₂}⁺151 (100); EI-MS: {M} 336 (100), {C₉H₁₁O₂} 151 (50).

Demethylation of 2-hydroxy-1-(3,4-dimethoxyphenyl)dodecan-3-one can becarried out by using BBr₃ as a reagent to produce a final product asshown in reaction scheme below. The synthetic procedure will generallyfollow a published method by which 1 mole of BBr₃ may be used todemethylate approximate 3 moles of2-hydroxy-1-(3,4-dimethoxyphenyl)dodecan-3-one at room temperature(Bhatt and Kulkarni, 1983). This may result in three products as shownin the reaction scheme below.

Preparation of 1-(3,4-dimethoxyphenyl)dodecan-2-ol

To a Grignard solution of decylmagnesiumbromide, prepared from Mg (0.05g) and 1-bromodecane (0.36 g), in THF (5 ml) under N₂ was added3,4-dimethoxyphenylacetal (0.5 g) in THF (5 ml). The mixture was stirredat room temperature for 5 hrs. Cold water (10 ml) was added following bythe addition of H₂SO₄ (1 M, 40 ml). The mixture was extracted twice withdiethylether (50 ml), washed with brine solution and the solventevaporated to give a liquid which was purified by column chromatographyto afford a colourless solid (yield 60%).

¹H-NMR: δ6.74-6.82 (3H,m), 3.88 (3H, s), 3.86 (3H, s), 3.78 (1H, m),2.77 (1H, m), 2.57 (1H, m), 1.52 (2H, m), 1.26 (16H, m), 0.88 (3H, t,b). ¹³C-NMR: δ14.17, 22.73, 25.84, 29.83, 29.67 (3C), 29.74, 31.96,36.88, 43.66, 55.88, 55.96, 72.75, 111.37, 112.58, 121.35, 131.15,147.71, 148.99. CI-MS: {M+1}⁺ 323 (25), {M+1−H₂O}⁺ 305 (100), {C₉H₁₁O₂}⁺151 (15); EI-MS: {M} 322 (100), {C₉H₁₂O₂} 152 (50).

Demethylation can be carried out as described above for2-hydroxy-1-(3,4-dimethoxyphenyl)dodecan-3-one. This may also result inthree products.

Preparation of 2-hydroxy-1-(3,4-dimethoxyphenyl)undecan-4-one

The title compound was prepared as described in the preparation of3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-5-one (page 34).

¹H-NMR: δ6.74-6.82 (3H,m), 4.26 (1H, m), 3.87 (3H, s), 3.86 (3H, s),2.62-2.84 (2H, m), 2.37-2.57 (4H, m), 1.55 (2H, m), 1.25 (8H, m), 0.87(3H, t, b). ¹³C-NMR: δ14.1, 22.63, 23.59, 29.07, 29.14, 31.68, 42.51,43.75, 48.14, 55.9, 55.94, 68.85, 111.29, 112.55, 121.37, 130.48,147.78, 148.95. CI-MS: {M+1}⁺ 323 (10), {M+1−H₂O}⁺ 305 (40), {C₁₀H₁₃O₃}⁺181 (80), {C₉H₁₉O}⁺ 143 (85); EI-MS: {M} 322 (100), {M−H₂O} 304 (90),{C₁₁H₁₃O₂} 177 (60), {C₉H₁₁O₂} 151 (50).

Demethylation can be carried out as described above for2-hydroxy-1-(3,4-dimethoxyphenyl)dodecan-3-one. This may also result inthree products.

A. Sarcoplasmic Reticulum Ca²⁺-ATPase Assay

SR membrane (75 μg/ml): 24 μl

Test substance: appropriate volumes to give a dose-effect concentration

Incubation buffer: up to 240 μl

A portion at each concentration (54 μl) was aliquoted into 4 designatedwells of the microplate (two of four wells were controls). Each well wasmixed with ATP solution (20 mM, 6 μl), except the controls, using an 8channel pipette. The controls were assayed in the absence of ATP orcalcium.

The plate was incubated with the lid on, for 30 min at 37° C., colourreagent (160 μl) was then added and mixed with citrate solution (34%, 30μl). The plate was developed at room temperature for 30 min thenabsorbance at 655 nm was read from the microplate reader. TheCa²⁺-ATPase activity was quantitated, from a P_(i) standard curve, asconcentration of liberated inorganic phosphate.

Stock solutions (10 mM) of test substance were prepared in DMSO, thenseries dilutions were made either in DMSO or in 1M HEPES to produce thedose-response concentrations. The maximum concentration of DMSO in thefinal assay solution was 2.5%.

Each concentration of test substance was assayed in duplicate in thepresence and absence of ATP or calcium. Phosphate standards are run oneach plate as follows:

P₁ (nmoles/60 μl) 0 1 2 3 4 6 8 10 P₁ stock (μl) 0 6 12 18 24 36 48 60H₂O (μl) 60 54 48 42 36 24 12 0

B. Preparation of SR Ca²⁺-ATPase Incubation Buffer

Buffer Stock Volume concentration conc. taken 0.1 mM KCl 2 M 5 ml 4 mMMgCl 1 M 0.4 ml 0.1 M Sucrose 2 M 5 ml 5 mM NaN₃ 1 M 0.5 ml 20 mMImidazole 1 M 2 ml H₂O to 100 ml

pH was adjusted to ˜7.4

C. Preparation of SR ATP Solution (10×)

Final 10× Stock Amount conc. conc. conc. taken 2 mM ATP 20 mM 0.126 g/10ml 66 μM CaCl₂ 0.66 mM 0.1 M 66 μl 30 μM EGTA 0.3 mM 0.1 M 30 μl SRbuffer to 10 ml

pH was adjusted to ˜7.4

D. Preparation of AMT Solution

3 parts of malachite green (0.05%)

1 part ammonium molybdate solution

The mixture was stirred at room temperature for 1 hour then Tween 20 (60μl per 100 ml) was added and stirred for ½ hour at room temperature.

3.2.5 Organ Bath Assay

Male guinea pigs, 3-4 weeks old, were killed by rapid cervicaldislocation without induced anaesthesia. Then, the guinea pigs weredissected to isolate the atria which were immediately mounted verticallyin an organ bath containing Krebs-Henseleit solution oxygenated withcarbogen.

One gram tension was applied to the atria and the base line continuouslyadjusted until it was stable for 20 mins. The rate and force ofcontraction were recorded using Mac Lab equipment.

Test substances in DMSO were assayed to a maximum concentration of 50 μMat a final concentration of 2.5% of DMSO.

Krebs-Henseleit Solution

1 liter I. MgSO₄.7H₂O 0.29 g NaCl 6.92 g KCl 0.35 g KH₂PO₄ 0.165 gD-glucose 2.10 g II. NaHCO₃ 2.10 g III. CaCl₂.2H₂O 1 ml (0.373 g/ml)

(I) was dissolved in an appropriate volume of phosphate free water,followed by the addition of (II) until all dissolved, then (III).

Results—SR Ca²⁺-ATPase Activity

1- (hydroxy- 3-methoxy- Concentration [6]- [8]- [8]- phenyl) (μM)gingerol gingerol dehydro dodecan-3-ol  0 100 100 100 100  1 121 139  93—  5 120 158  88 102  10 117 165  92 108  25 137 200  95 140  50 160 139 65 101 100 — — —  98 200 — — —  16 Con- 3-hydroxy-1- 3-hydroxy-1- cen-1-(4-hydroxy-3- (4-hydroxy-3- (4-hydroxy-3- tration methoxyphenyl)methoxyphenyl) methoxyphenyl) (μM) dodecan-5-one; decan-1-onedodecan-1-one  0 100 100 100  1 — 104 122  5 113 152 111  10 136 174 133 25 158 195 147  50 126 180 158 100  87 222 149 200  18 — —

Effect of gingerols and their derivatives on the Ca²⁺-ATPase of dogcardiac SR.

Results

Table 1: SR Ca²⁺-ATPase Activity and Inotropic Activity of Gingerols andGingerol Analogues. IC₅₀=conc. for 50% Inhibition.

SR Ca²⁺- FORCE OF HEART COMPOUNDS ATPase (μM) CONTRACT^(n) RATE[6]-gingerol ↑60% @ 50 ↑86% @ 50 ↑10% @ 50 IC₅₀ > 100 [8]-gingerol ↑100%@ 25 ↑160% @ 3 ↑31% @ 3 IC₅₀˜100 [8]-dehydrogingerol ↓35% @ 50 ↑80% @ 25↑17% @ 25 IC₅₀˜50 [8]-isogingerol ↑64% @ 50 inactive inactive IC₅₀ > 100[8]-orthogingerol ↑40% @ 100 inactive inactive IC₅₀ > 100[6]-demethoxygingerol IC₅₀ > 100 inactive inactive [8]-demethoxygingerol↑60% @ 50 inactive inactive IC₅₀ > 1003-hydroxy-1-(4-hydroxy-3-methoxyphenyl) IC₅₀ > 100 ↑130% @ 10 ↑30% @ 10dodecan-5-one (3.90) ↑270% @ 5 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)↑95% @ 25 ↓11% @ 10 ↓5% @ 10 decan-1-one (3.92) IC₅₀˜50 ↓13% @ 303-hydroxy-1-(4-hydroxy-3-methoxyphenyl) ↑60% @ 10 ↑11% @ 30dodecan-1-one (3.91) IC₅₀˜50 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)↑40% @ 25 ↑108% @ 10 ↑50% @ 10 dodecane IC₅₀ 50-1005-hydroxy-1-(4-hydroxy-3-methoxyphenyl) ↑57% @ 25 ↑79% @ 30 ↑weaklydodecane (3.94) IC₅₀˜25 1-(4-hydroxy-3-methoxyphenyl)dodecan-3- inactiveinactive one [8]-gingerdiol ↑54% @ 50 inactive inactive IC₅₀ > 100

inactive bisgingerol inactive inactive

↑increase or stimulation ↓decrease or inhibition

Guinea pig atria testing on a range of phenolic substances revealed anumber of interesting substances, including gingerols shown in table 1,that give rise to increased contractility of the atria. [8]-Gingerolappeared to be one of the most potent cardiotonic substances in theseries. All the test substances increased the heart rate significantly.It was observed that all gingerol derivatives except [8]-dehydrogingeroldeveloped maximum tension rapidly in a min or so after addition of adose. In contrast, [8]-dehydrogingerol and digoxin took a few mins fortension to reach maximum. Digoxin was observed to cause arrhythmia a fewmins after addition of a dose.

Discussion

Substances of the gingerol series may exhibit similar mechanisms ofaction to those described for fatty acids but with selectivity towardsSR Ca²⁺-ATPase only. They produced in general a biphasic activityprofile. They stimulate the ATPase at low concentration but weaklyinhibit the enzyme at high concentration. SAR of gingerol revealed aunique aromatic feature which is essential for cardiotonic activity.[8]-Gingerol appeared the most potent cardiotonic substance in theseries, however, it is readily chemically (Mustafa et al., 1993) andbiochemically (Young-Joon, 1994) degraded even in its pure state. Uponstorage for a long time or exposure to an acidic environment,dehydration occurs rapidly particular at low pH to produce a shogaolwhich is devoid of cardiotonic activity. This could be a reason for theapparent short half life of [8]-gingerol when it was given to dogs (0.3mg/kg, i.v.) resulting in increased cardiac contractility of about 30%for 10 min (Mitsubishi Co, 1982).

Some analogues such as 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol,3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-1-one and3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-1-one are probablychemically and biochemically much more stable than gingerols. Therefore,further investigation of the physicochemical properties as well asmechanism(s) of action of these substances, including gingerols, isrequired in relation to cardiotonicity. The mode of action of gingerolsas cardiotonic agents has been thoroughly investigated and it wasproposed that they act by direct stimulation of the cardiac SRCa²⁺-ATPase. This may load extra calcium into the intracellular storesin the sarcoplasmic reticulum allowing more calcium to be released onstimulation of the heart resulting in increased cardiac contractility.However, this study has indicated that the positive cardiotonicity ofgingerols may not be simply related directly to its activation of SRCa²⁺-ATPase. It was found that [8]-dehydrogingerol, a SR Ca²⁺-ATPaseinhibitor, also produced a cardiotonic effect on guinea pig atria,however, the maximum effect was much delayed compared to [8]-gingerolwhich rapidly produced increased cardiac contractility 15 sec afteraddition of the drug. This indicated that [8]-gingerol may exert actionsoutside of the cell in addition to activation of SR Ca²⁺-ATPase. Thecardiotonic action of 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol [3.93]was confirmed from independent studies, carried out at our request aspart of our agreement with Johnson & Johnson Research Ltd (J&J), byProf. James Angus from the University of Melbourne. Preliminarymechanistic studies, shown in the table below, of the substance revealedthat the positive inotropic activity (1×10⁻⁶ to 3×10⁻⁵ inconcentrations) of the substance was not due to sympathetic nervestimulation nor mediated by α- or β-adrenoceptors. It had neither aneffect on the neuromuscular junction from phrenic nerve stimulation ofrat diaphragm, nor an effect on sympathetic neuro-effector function inthe rat vas deferens. A tachycardial effect of the substance on guineapig atria was, however, observed at significant rate, 50-60% ofisoprenaline E_(max) and the response was not α- or β-adrenoceptormediated. The mechanism of tachycardia and positive inotropic responseof the substance was shown to be due to the release of neuropeptides andprobably cGRP and indirectly a release of histamine since pretreatmentof the guinea pig atria with capsaicin or capsazepine, a capsaicinantagonist, abolished inotropic effect of the test substance. Similarlycapsaicin was observed to have tachycardial and inotropic effects onguinea pig atria and these effects were blocked in the presence ofeither 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol or capsazepine.

Cardiovascular and Neuro-effector Junction Activities of GingerolAnalogues

right atrium- right left vas vagus atrium atrium deferens rat rat ratrat 3-hydroxy- some no change no change no effect 1-(4- enhanced basalrate 10⁻⁷- 10⁻⁴ M hydroxy-3- slowing 10⁻⁷-10⁻⁵ M 3 × 10⁻⁵ M methoxy-10⁻⁶-10⁻⁵ M (sharp phenyl) fall decan-1- 10⁻⁴ M) one 1-(4- dramatictachycardia no change ↓30% hydroxy-3- enhancement 3 × 10⁻⁷- in force10⁻⁵ M methoxy- n = 1 3 × 10⁻⁶ M 10⁻⁷-10⁻⁵ M (vehicie?) phenyl)10⁻⁶-10⁻⁵ M (with (n = 2) dodecan-3-ol propranolol present) rightatrium- right left diaphragm vagus atrium atrium rat guinea pig guineapig guinea pig 3-hydroxy- no effect weak? no change no 1-(4- 10⁻⁵ Menhanced basal rate inotropism hydroxy-3- ↓10% slowing 10⁻⁷-10⁻⁵ M noeffect methoxy- 3 × 10⁻⁵ M on isopre- phenyl) naline decan-1- responseone (up to 30 μM) 1-(4- ↓10% no tachycardia inotropic hydroxy-3- 10⁻⁵ Menhancement 10⁻⁷- 10⁻⁶- methoxy- (vehicle?) of vagal 3 × 10⁻⁵ M 3 × 10⁻⁵M phenyl) slowing to 60% of ˜20% dodecan-3-ol 10⁻⁷-10⁻⁵ M isopren.isopren. Emax max. (not (not blocked by in rat) praz., prop.)

Cardiac Electrophysiological Activities of Gingerol and its Analogue

The basic cardiac electrophysiological properties were assessed usingcardiac Purkinje fibres obtained from adult mongrel dogs.

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decanone (n=3)

Action Action Potential Potential Action Maximum Duration DurationConcen- Potential rate of 50% 90% tration Amplitude depolarisationrepolarisation repolarisation (μM) (mV) (V/s) (ms) (ms)  0 126 ± 4 605 ±70 200 ± 20 283 ± 22  5 125 ± 3 524 ± 66 176 ± 17 270 ± 26 10 127 ± 4527 ± 78 157 ± 23 257 ± 25 20 123 ± 6 521 ± 87 124 ± 16 245 ± 17 30 115± 7 423 ± 45  94 ± 10 212 ± 21 40 117 ± 7 442 ± 61  81 ± 10 201 ± 13 50110 ± 9 363 ± 74 55 ± 4 188 ± 19

[8]-gingerol (n=9)

Action Action Potential Potential Action Maximum Duration DurationConcen- Potential rate of 50% 90% tration Amplitude depolarisationrepolarisation repolarisation (μM) (mV) (V/s) (ms) (ms)  0 123 ± 2 576 ±71 278 ± 18 385 ± 24  5 124 ± 2 532 ± 42 242 ± 20 363 ± 27 10 120 ± 4483 ± 41 207 ± 20 339 ± 26 20 119 ± 2 499 ± 41 142 ± 20 285 ± 26 30 114± 3 443 ± 45 107 ± 17 248 ± 25 40 116 ± 4 516 ± 67  88 ± 15 229 ± 22 50109 ± 8 471 ± 73  76 ± 12 221 ± 19

Electrophysiology Results

Electrophysiology was carried out on dog heart purkinje fibre to testfor potential arrhythmia. [8]-Gingerol and an analogue 3.92, up to 10μM, caused insignificant effect on the rate of depolarization, amplitudeof action potential and duration of action potential at 50% and 90%repolarization. At high concentration (50 μM), however, [8]-gingerolreduced approximate 80% and 50% of action potential duration at 50% and90% repolarisations respectively. Whereas the analogue reduced 75% and30% respectively.

Vasodilation of Gingerol Analogues on Rat Mesenteric Artery

Small arteries (200-300 μm in diameter) isolated from rat mesentery wereprecontracted with endothelin prior to addition of the test substancesand tested for relaxation.

Results EC₅₀ Compounds (μM) 1-(4-hydroxy-3- 0.1 methoxyphenyl)dodecan-3-ol enantiomer-1 0.5 enantiomer-2 0.32 5-hydroxy-1-(3,4- 0.69methylene- dioxyphenyl) dodecan-3-one [8]-paradol 0.05 3-hydroxy-1-(4-0.04 hydroxy-3- methoxyphenyl) dodecan-5-one

At 10⁻⁷−10⁻⁶ M, these substances gave complete relaxation ofprecontracted blood vessels and the relaxation was probably due to therelease of neuropeptide from sensory nerves, however the identity of thepeptides responsible for the relaxation is not certain, as yet. Therelaxation was abolished by pretreatment with capsaicin andalternatively, pretreament of the gingerol analogues also abolished thevasodilation by capsaicin. Preliminary studies on cultured cells fromrat dorsal root ganglion have shown that1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol increased intracellularcalcium, which is probably a mechanism of action of the gingerolanalogues. The rise in intracellular calcium may result in release ofneuropeptide(s) from sensory nerves that cause vasodilation of ratmesenteric artery. The exact receptor(s) where gingerol analogues exerttheir actions remains to be defined. Further investigation is inprogress.

Platelet Aggregation Activity of Gingerol Analogues

Blood was collected from healthy volunteers who had taken no medicationin the previous two weeks. The anti-coagulant used was 3.8% trisodiumcitrate. Platelet rich plasma was prepared and incubated with[³H]-serotonin. This was followed by addition of gingerol analogues.Platelet activation was initiated using the EC₅₀ concentration forarachidonic acid. The percentage of [³H]-serotonin release was measuredusing a liquid scintillation counter. Then, dose-response curves wereestablished and IC₅₀ values were obtained.

Results Compounds IC₅₀ (μM) Aspirin 23.4 ± 1.7 [6]-gingerol 73.8 ± 6.6[8]-gingerol 70.4 ± 3.8 1-(4-hydroxy-3-methoxyphenyl) 58.0 ± 1.9dodecan-3-ol [8]-gingerdiol 82.6 ± 9.0 3-methyl-1-(4-hydroxy-3- 45.3 ±1.6 methoxyphenyl)undecan-3-ol 3-methyl-1-(4-hydroxy-3- 75.3 ± 3.1methoxyphenyl)tridecan-3-ol 1-hydroxy-1-(4-hydroxy-3- 69.4 ± 2.6methoxyphenyl)undecan-2-one

Neurokinin Activity of Gingerol Analogues

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane [3.93], was tested onguinea pig submaxillary membrane for neurokinin-1 (NK-1) antagonistactivity with tritium labelled [³H] Substance P and found to haverelatively potent inhibition of the binding of Substance P to NK-1receptor. The substance exhibited a dose-dependent inhibition ofSubstance P on NK-1 receptor. It gave approximately 80% inhibition at 30μM.

Lipoxygenase Activity of Gingerol Analogues

3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane [3.93] was tested at MDSPANLABS: Pharmacology Services (Taiwan) for 5-lipoxygenase activity fromrat basophilic leukemia cells (RBL-1) with arachidonic acid assubstrate. The inhibitory activity of the substance was quantitated,using radioimmunoassay, from the formation of 5-HETE. At 10 μM, thesubstance gave approximate 90% inhibition of 5-lipoxygenase activity.

Toxicity—Brine Shrimp Assay

The brine shrimp assay procedure determines LC₅₀ values of activecompounds. Activities of a broad range of known active compounds aremanifested as toxicity to brine shrimp (Artemia salina Leach). There aremany applications of the assay including analysis of toxic substances,anaesthetics, morphine-like substances and cocarcinogenicity of phorbolesters. The assay shows good correlation with some cytotoxicities andits utility as a prescreen for some antitumour activities has beenrecently confirmed.

DMSO (dimethyl sulfoxide) was the solvent of choice because of its goodsolubilising properties and also because the substances used in theATPase inhibition assays were already prepared with DMSO.

The method for testing solvent toxicity used was basically that reportedby J L McLaughlin in Methods of Plant Biochemistry (1991), vol. 6 (KHostettman, ed.), Academic Press, London, 1-32. DMSO solutions of thesubstances to be tested were added directly to the vials containing thebrine shrimp. As the concentration of DMSO is that we wished to use washigher than the recommended 1% v/v testing of the toxicity of the DMSOwas therefore necessary. The concentrations of DMSO tested on theshrimp, along with the results from the assay which was done induplicate are listed below.

Concentrations of DMSO Tested

Conc (% v/v) % Deaths 0 0 1 0 2 0 3 0 4 0 5 9 7 12 9 18 11 57 13 96 15100 20 100 25 100

Bioassay

Brine shrimp toxicity was assayed, except for some minor modifications,according to the method of McLaughlin et al as reported in Brine Shrimp:A convenient bioassay for active plant constituents, B N Meyer, N RFerrigni, J E Putnam, L B Jacobsen, D E Nichols and J L McLaughlin inPlanta Medica (1982), 45, 31-34 and Crown gall tumours on potato discsand brine shrimp lethality: Two simple bioassays for higher plantscreening and fractionation. J L McLaughlin. Methods of PlantBiochemistry (1991), vol. 6 (K Hostettman, ed.), Academic Press, London,1-32. Ten shrimp were transferred to each of the vials and the volumeadjusted to 4.9 mL. Each dose was performed in triplicate, including thecontrol. In quick succession, the appropriate volume of additional DMSOfor each dose, required to achieve a final concentration of 2%, wasadded before the appropriate volume of test solution. The vials weregently mixed and the time noted. After 24 hours, the number of survivorswere counted and % mortality was determined. The test compounds wereassayed at concentrations of 100 μM, 25 μM, 5 μM, 1 μM and 0.2 μM (andwhere appropriate concentrations of 0.04 μM and 0.008 μM).

The brine shrimp were able to survive without food in the vials over the24 hour period and were therefore not fed.

The dose-response curves were constructed using the Sigmaplot computerprogram and the LC₅₀ value was calculated from the intersection point ofthe curve and the 50% mortality line. The LC₅₀ values were expressed inboth μM and μg/mL.

Table 2: LC₅₀ Values of Phenolic Substances from Brine Shrimp Bioassay

For substances with low toxicity, the greater sign “>” indicated thehighest concentration at which the assay was carried out asprecipitation of the substances occurred above that concentration.

LC₅₀ MWt Compound μM μg/ml 294 [6]-gingerol >100 >29 322 [8]-gingerol 6421 322 [8]-orthogingerol 9.6 3.1 322 [8]-isogingerol 11 3.5 3223-hydroxy-1-(4-hydroxy-3- 67 22 methoxyphenyl)dodecan-5-one 2943-hydroxy-1-(4-hydroxy-3- 66 19 methoxyphenyl)decan-1-one 3223-hydroxy-1-(4-hydroxy-3- 10 3.2 methoxyphenyl)dodecan-1-one 320[8]-dehydrogingerol 41 13 2645-hydroxy-1-(4-hydroxyphenyl)decan-3-one >100 >26 2925-hydroxy-1-(4-hydroxyphenyl)dodecan-3-one 25 7.3 2905-hydroxy-1-(4-hydroxyphenyl)dodecan-1- 2.6 0.75 ene-3-one 3081-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol >100 >31 3081-(4-hydroxy-3-methoxyphenyl)dodecan-5-ol 6.6 2.0 3021-(4-hydroxy-3-methoxyphenyl)dodecane- 5.4 1.6 1,4-diene-3-one 3205-hydroxy-1-(3,4- 3.0 0.96 methylenedioxyphenyl)dodecan-3-one 414podophyllotoxin 3.8 1.6 (5.8)* (2.4) *LC₅₀ values determined by Meyer etal. (1982)

Discussion

In this bioassay, the estimated LC₅₀ value of the test compoundindicates its toxicity to brine shrimp. A more useful comparison ofpotencies can be obtained by looking at the μM instead of μg/mLconcentrations in Table 2. Podophyllotoxin was tested in order to checkwhether the bioassay's results were comparable with those of Meyer etal. The LC₅₀ from this study was 3.8 μM and is reasonably close to theLC₅₀ value of 5.8 μM determined by Meyer et al.

Effect of Gingerol Analogues Towards Rat Dorsal Root Ganglia

Capsaicin, the pungent component in peppers of the Capsicum genus,family Solanaceae, has the ability to excite a subset of sensoryneurons, which include polymodal nociceptors and warm thermoceptors(Fitzgerald, 1983) by opening non-selective cation channels that arepermeable to Na⁺, K⁺, and Ca²⁺ (Bevan and Szolcsanyi, 1990). It has beenshown that capsaicin evoked a concentration-dependent rise in [Ca^(2+])_(i) in cultured dorsal root ganglion neurons. The duration of theelevation of [Ca²⁺]_(i) depended on the concentration of capsaicin(Choleswinski, et al., 1993). In this investigation peak [Ca²⁺]_(i)transient measurements are used as a model for testing gingerolanalogues.

EXPERIMENTAL

DRG from neonatal (3-5 days old) rat or adult Sprague-Dawley rats wereincubated in Hanks CMF saline with 0.05% collagenase and 0.25% trypsinfor 25 min at 37° C. Individual cells were obtained by trituration withfire polished Pasteur pipettes of diminishing diameters. Neurones wereisolated from the cell suspension in 30% Percoll and then plated oncollagen coated coverslips or in 24-well plates, then cultured inneurobasal medium with B27 supplement, 50 ng/ml 2.5 S nerve growthfactor, 2 mM 1-glutamine and 100 U/ml penicillin/streptomycin. Cultureswere maintained at 37° C. with 5% CO₂. 30% Percoll increases thepercentage of capsaicin-sensitive DRG neurones up to 70%. For peak[Ca²⁺]_(i) transients measurements, cells on coverslips were incubatedwith 5 μM Fura-2 AM for 30 min at 37° C. The coverslips were thenmounted on a chamber attached to a fast sample application perfusionsystem which allows changing solutions in the second range. Recordingswere made on the stage of a Nikon Diaphot inverted microscope fittedwith a Nikon 40×Fluo (NA 0.85) DL Ph3 or 40×Fluo (NA 1.3) oil objective.[Ca²⁺]_(i) was calculated from dual excitation wavelength (340/380 nm)fluorescence measurements following an intracellular calibrationprocedure by the Grynkiewicz equation, using MCID M2/M4 v.3.0 (ImagingRes. Inc.) software. Cells were continuously perfused with a solutionconsisting of 140 mM NaCl, 2 mM CaCl₂, 5 mM KCl, 20 mM HEPES, 10 mMglucose, pH 7.4. To study KCl evoked depolarisation, 50 mM NaCl wasreplaced by equimolar KCl. Cytoplasmic localisation of Fura-2 was testedwith the Mn²⁺ quenching technique (Dedov and Roufogalis, 1998). Allexperiments were performed at room temperature (20-23° C.).

Results

Typical changes in [Ca²⁺]_(i) upon depolarisation were evoked by 50 mMKCl and by application of 1 μM capsaicin; both were applied for 30 sec.Capsaicin-evoked peak [Ca²⁺]_(i) transients are characterised by a fastrise and long-steady state [Ca²⁺]_(i) clearance from the cytoplasm. Incapsaicin-sensitive cells the half-time of cytoplasmic Ca²⁺ clearancewas proportional to the amplitudes of peak [Ca²⁺]_(i) transients.

To examine the effect of gingerol derivatives, one or several compoundsin succession at a concentration of 10 μM were applied for 1 min to theDRG neuronal cells followed by washing out for 4 min with physiologicalsolution. To the capsaicin-sensitive cells, 10 μM capsaicin and 50 mMKCl were applied, respectively, to confirm the viability of the cells.In addition, morphological appearances of the cells were also examinedat the end of experiment. All experiments were carried out in thepresence of 2 mM Ca²⁺.

Effect of the Gingerol Analogues and Capsaicin in Evoking [Ca²⁺]_(i)Transients in DRG Neuronal Cells in Culture

Number of capsaicin- Peak [Ca²⁺]_(i) Name of sensitive cells (nM)compounds responding (Average ± SD) Capsaicin  5 824 ± 122 (highresponses) 10 134 ± 78 (low responses) 1-(4-hydroxy-3-  5 661 ± 376methoxyphenyl) (high responses) dodecan-3-ol 10 187 ± 66 (low responses)3-hydroxy-1-(4-  5 167 ± 105 hydroxy-3- methoxyphenyl) dodecan-5-one5-hydroxy-1-(4-  7 279 ± 111 hydroxy-3- methoxyphenyl) dodecan-3-one5-hydroxy-1-(3-  2 281 ± 42 hydroxy-4- methoxyphenyl) dodecan-3-one5-hydroxy-1-(2-  2 245 ± 64 hydroxy-3- methoxyphenyl) dodecan-3-one5-hydroxy-1-(4-  2 243 ± 71 hydroxyphenyl) dodecan-3-one5-hydroxy-1-3,4-  2 225 ± 78 methylenedioxyphenyl) dodecan-3-one1-(4-hydroxy-3-  2 246 ± 35 methoxyphenyl) dodecane-3,5-diol2-hydroxy-1-(4-  2 215 ± 7 hydroxy-3- methoxyphenyl) undecan-1-one[8]-shogaol  2 110 ± 56 [8]-paradol  1 200 3-hydroxy-1-(4-  0 out of 5 0 hydroxy-3- methoxyphenyl) decan-1-one 3-methyl-1-(4-  0 out of 4  0hydroxy-3- methoxyphenyl) undecan-3-ol 1-(4-hydroxy-3-  0 out of 5  0methoxyphenyl) dodecan-5-ol

All gingerol derivatives at 10 μM, except the last three compounds,evoked [Ca²⁺]_(i) transients in capsaicin-sensitive cells.

No capsaicin-insensitive DRG neuronal cells responded to the gingerolderivatives. There are some preliminary indications that gingerolderivative evoked [Ca²⁺]_(i) transients have a faster [Ca²⁺]_(i)clearance from the cytoplasm, compared to the slow decay of [Ca²⁺]_(i)from capsaicin evoked [Ca^(2+]) _(i) transients. Either differentaffinities of these compounds for the receptor in comparison tocapsaicin, or interaction with sub-populations/classes of receptors oradditional stimulation of [Ca²⁺]_(i) efflux from the cells were proposedto account for these differences. Both capsaicin and1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol evoked [Ca²⁺]_(i) transientswere abolished or greatly diminished in Ca²⁺-free medium or on theapplication of ruthenium red (1 μM), a non-specific capsaicinantagonist.

Conclusion

1. The data obtained suggest that 11 (out of the 14 gingerol derivativestested) may bind to capsaicin-receptor in DRG neuronal cells in culture,subsequently opening ion channel(s) which are permeable to extracellularCa²⁺. The rise in intracellular Ca²⁺ in cells is known to mediate manybiological events, particularly the signal transduction pathway whichmay lead to release of neuropeptides or factors that subsequentlymodulate pain transmission mechanisms. A rise in intracellular Ca²⁺ isalso known for capsaicin to result in desensitisation of nerve fibrestoward further firing from pain stimuli.

2. There is structural specificity, particularly at the hydroxy moietyon the side chain, to evoke [Ca²⁺]_(i) transients in DRG neuronal cellsfrom the gingerol derivatives. Despite the very close structuralresemblance between 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol and3-methyl-1-(4-hydroxy-3-methoxyphenyl)undecan-3-ol, the former showed asignificant rise in [Ca²⁺]_(i), whereas the latter was ineffective.These data will direct future synthesis of more effective gingerolderivatives.

3. The difference in [Ca²⁺]_(i) clearance from cytoplasm betweencapsaicin and gingerol derivatives may make the latter less neurotoxicthan capsaicin because neurotoxicity is Ca²⁺ dependent (Caterina et: al,1997)

Cyclo-oxygenase Assay

Cyclooxygenase (COX) is a haemprotein which catalyses the formation ofPGH₂ from arachidonic acid. Two isoforms of COX have been-identified anddesignated as COX-1 and COX-2. COX-1 is constitutively expressed in mosttissue and performs a “housekeeping” function to synthesiseprostaglandins which regulate normal cell activities includingantithrombogenic activity and cytoprotection of gastric mucosa andkidney. COX-2 is an inducible enzyme which responds more rapidly andtransiently to mediators of immunity, inflammation and tissue repair.Recently attention has been paid to the activity of COX-2 with increasedevidence that downregulation of this enzyme activity will be importantin control of inflammation and pain and an important strategy forpreventing cancer since the enzyme catalyses the formation ofprostaglandins which respectively mediate inflammation, pain, and havemultiple effects that favour tumorigenesis. Selective inhibition ofCOX-2 will have many therapeutic applications without causing manyundesirable effects to normal cell function.

Cyclo-oxygenase (COX) assay was performed with cells in culture preparedfrom rat basophilic leukemia (RBL) 2H3 cell lines. Cells were culturedin EMEM containing 10% fetal calf serum and antibiotic until cellsreached confluence. Harvested cells were subsequently seeded on 24-wellplates at 1×10⁶ cells/ml then incubated at 37° C. for 3 hours. Cellswere washed twice with incubation buffer (1.5 ml) containing 5 mM Hepes,140 mM NaCl, 5 mM KCl, 0.6 mM MgCl₂, 1 mM CaCl₂ and 55 mM glucose. Cellswere then covered with 0.49 ml of buffer, followed by addition ofsamples/solvents (0.005 ml) and incubated at 37° C. for 5 min on anorbital shaker. Arachidonic acid (0.005 ml, containing 50% EtOH) wassubsequently added, and the plate was incubated at similar conditionsfor a further 10 min. The supernatant (0.1 ml) was aliquoted formethoxime derivatisation of PGD₂, which was carried out by heating thesupernatant with methoxime solution (1:1) at 60° C. for 30 min accordingto the instructions provided with the kit. The resultant solution wasdiluted with EIA buffer and stored on ice for EIA, following theprotocol provided with the kit. Validation of COX enzyme activity wascarried out using EIA in which the enzyme activity was characterisedagainst various concentrations of AA and a time course of enzymeactivity. All compounds were dissolved in DMSO and assayed at a finalconcentration of 10 μM. Indomethacin was used as reference compound. Theconcentration of DMSO in the assay was maintained at 1.5%.

Lipoxygenase Assay

Lipoxygenases, including 5-, 12-, and 15-lipoxygenases and theirproducts play enormous roles in maintaining cellular function. However,they are also the key factors that cause many pathophysiologicalconditions. Inhibition of these enzymes hence has many therapeuticapplications in the treatment of inflammatory, allergic, cardiovascularand skin diseases.

Among these enzymes, 5-lipoxygenase and their products, particularly theleukotriene series, are the most important and extensively studied,revealing that the enzyme and its products mediate certain respiratory,cardiovascular, renal, gastrointestinal, and CNS disorders. Theprincipal therapeutic targets for 5-lipoxygenase inhibitors includeallergic diseases, in particular, human bronchial asthma; chronicinflammatory diseases; myocardial ischemia; and inflammatory associatedpain.

Lipoxygenase (LP) assay was performed with cell-free enzyme (Wong et al,1991) prepared from rat basophilic leukemia (RBL) 2H3 cell lines. Cellswere cultured in EMEM containing 10% fetal calf serum and antibioticuntil cells reached confluence. Harvested cells were resuspended inHepes (10 mM) buffer, pH 7.4, containing 1 mM EDTA at 1×10⁷ cells/ml,and disrupted by nitrogen cavitation using a Parr bomb at 750 psi for 15min. The broken cells were centrifuged at 15,000 g for 30 min. Aliquots(0.1 ml) of the supernatant were preincubated with or without drugs inHepes buffer (10 mM Hepes, pH 7, 1 mM EDTA and 150 mM NaCl) for 5 min,and the reaction was initiated with the addition of 50 μl of CaCl₂ (16mM), 50 μl of ATP (2 mM), 5 μl of PAF (2.5 mg/ml) and 5 μl of AA (2.5mM). The reaction mixture (1 ml in total) was incubated at roomtemperature for a further 8 min, then diluted with EIA buffer at 1/200and 1/2000 dilutions and stored on ice for EIA following the protocolprovided with the kit. Validation of LP enzyme activity was carried outby a UV spectrophotometric method in which the enzyme activity wascharacterised against various concentrations of Ca²⁺, ATP, PAF and AAwith the measurement of the formation of diene conjugated products of LPat 235 nm.

All compounds were dissolved in DMSO and assayed at a finalconcentration of 10 μM. NDGA was used as reference compound. Theconcentration of DMSO in the assay was maintained at 1.5%.

Results

Cyclo- Lipoxy- oxygenase genase activity activity Name of compounds %activity % activity 1-(4-hydroxy-3- 9 31 methoxyphenyl)dodecan- 3-ol3-hydroxy-1-(4- 18 193 hydroxy-3- methoxyphenyl)dodecan- 5-one5-hydroxy-1-(4- 16 65.5 hydroxy-3- methoxyphenyl)dodecan- 3-one5-hydroxy-1-(4- 55 202 hydroxy-3- methoxyphenyl)decan-3- one5-hydroxy-1-(4- 19 37 hydroxy-3- methoxyphenyl)dodecan- 1-ene-3-one5-hydroxy-1-(3- 23 8 hydroxy-4- methoxyphenyl)dodecan- 3-one5-hydroxy-1-(2- 17 118 hydroxy-3- methoxyphenyl)dodecan- 3-one5-hydroxy-1-(4- 63 450 hydroxyphenyl)dodecan- 3-one 5-hydroxy-1-(3,4- 59107 methylenedioxyphenyl) dodecan-3-one 1-(4-hydroxy-3- 10 26methoxyphenyl)dodecane- 3,5-diol 1-hydroxy-1-(4- 16 83 hydroxy-3-methoxyphenyl) undecan-2-one 2-hydroxy-1-(4- 95 61 hydroxy-3-methoxyphenyl)undecan- 1-one [8]-shogaol 21 0 1-(4-hydroxy-3- 2methoxyphenyl) dodecane-1,4-diene-3-one [8]-paradol 11 813-hydroxy-1-(4- 123 175 hydroxy-3- methoxyphenyl) decan-1-one3-methyl-1-(4- 15 3 hydroxy-3- methoxyphenyl) undecan-3-ol3-methyl-1-(4- 5 hydroxy-3- methoxyphenyl) tridecan-3-ol 1-(4-hydroxy-3-12 35 methoxyphenyl) dodecan-5-ol Indomethacin (1 μM) 23 NDGA (0.5 μM)20

There are structure-specific activities of the gingerol derivatives ininhibition of cyclooxygenase (COX) and lipoxygenase (LP). It wasobserved that alteration of the aromatic moiety and/or the functionalgroup on the side chain of the gingerol derivatives severely altered theinhibitory activity of the compounds towards LP. This was, however, notthe case in the inhibition of COX. Double bonds and methyl branches onthe side chain seem to effectively enhance inhibitory potency of thegingerol derivatives towards lipoxygenase. These results in relation tothe inhibition of cyclo-oxygenase and lipoxygenase of the gingerolderivatives, particularly gingerols and shogaol, support the traditionaluse of ginger in the treatment of inflammatory diseases and associatedpain.

The effective amount of the active compound required for use in theabove conditions will vary both with the route of administration, thecondition under treatment and the host undergoing treatment, and isultimately at the discretion of the physician. In the above mentionedtreatments, it is preferable to present the active compound as apharmaceutical formulation. A pharmaceutical formulation of the presentinvention comprises the active compound together with one or morepharmaceutically acceptable carriers and optionally any othertherapeutic ingredient. The formulation may conveniently be prepared inunit dosage form and may be prepared according to conventionalpharmaceutical techniques. Additionally, the formulations may includeone or more accessory ingredients, such as diluents, buffers, flavouringagents, binders, disintegrants, surface active agents, thickeners,lubricants, preservatives, enteric coatings and the like.

Pharmaceutical Formulation

A typical tablet formulation comprises 20-50 mg of the activeconstituent, 50-200 mg of lactose, 5-30 mg of maize starch and 0.2-1 mgof magnesium stearate. Preferably, the tablet formulation comprises20-50 mg of the active constituent, about 100 mg of lactose, about 15 mgof maize starch and about 0.5 mg of magnesium stearate.

Modified Ginger Extract Formulation

The modified ginger extract can be administered in a liquid formula orsyrup formulation. A typical liquid formula comprises 50-500 mg ofextract in alcohol (max. 80% v/v) or glycerol, or in a sugar basepreparation (1:1 liquid to sugar ratio). Alternatively, modified gingerextract can be administered in a solid dosage form, which can be eitheras tablet, capsule, or powder. A typical tablet formulation comprises50-500 mg of the modified ginger extract, 5-30 mg of maize starch ormicrocrystalline cellulose and 0.2-1 mg of magnesium stearate.Preferably, the tablet formulation comprises 50-500 mg of the extract,about 15 mg of maize starch or cellulose and about 0.5 mg of magnesiumstearate. Capsule or powder dosage forms also contains 50-500 mg of themodified ginger extract. Enteric coatings to protect against degradationmay be desirable.

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The claims defining the invention are as follows:
 1. A compound offormula (I) or a pharmaceutically acceptable derivative thereof:

where R₁ is H, OH, OC₁₋₄alkyl, NO₂ R₂ is OH, OC₁₋₄alkyl, OC═OC₁₋₄alkylor OC═OPh where the Ph can be optionally substituted by halogen, C₁₋₃alkyl or NO₂; R₁ and R₂ along with the two carbon atoms of the phenylring to which they are attached can combine to form a 5 or 6 memberedheterocyclic ring comprising 1 or 2 heteroatoms selected from O, S or N;R₃ is C₂₋₁₂alkyl, C₂₋₁₂alkenyl or C₂₋₁₂alkynyl each optionallysubstituted by one or more substituents selected from —OR, ═O, nitro,halogen, —NRR′, —COOR or —CONRR′ where R and R′ are H or C₁₋₄alkyl; R₃may be a linking group of a bis compound where R₃ is C₂₋₁₂alkylene,C₂₋₁₂alkenylene or C₂₋₁₂alkynylene each optionally substituted by one ormore substituents selected from —OR, ═O, nitro, halogen, —NRR′, —COOR or—CONRR′ where R and R′ are H or C₁₋₄alkyl; R₄ is H, CH₃, OH or ═O; whenR₄ is ═O, then the carbon to which R₄ is attached is not bonded to H; Wis C(═O)—CH₂, CH═CH—, CH₂CO, CH(OH)—CH₂, C(CH₃)(OH)CH₂, CH₂CH(OH),CH₂C(CH₃)OH, CO, CHOH, C(CH₃)(OH), CH₂, CH₂CH₂; X is —CH—OH, C(CH₃) OH,CH₂, CH(CH₃) or —C═O; Y is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;provided that one of W, X or Y has an OH group and provided that when(1) R₁ is OC₁₋₄alkyl, R₂ is OH or OAcyl, W=CH₂CH₂ and X=C═O, R₃ is C₂₋₁₂alkyl, R₄ is H, then Y is not CHOH (gingerols); (2) R₁ is OCH₃, R₂ isOH, W is CH₂CH₂, R₃ is C₅ or C₇ alkyl, R₄ is H and X=CHOH then Y is notCHOH (gingerdiol); (3) R₁ is OCH₃, R₂ is OH, W is CH═CH, R₃ is C₂₋₁₂alkyl, R₄ is H and X is C═O, then Y is not CHOH (dehydrogingerols); (4)R₁ is OCH₃, R₂ is OH, W=CH₂CH₂, X is CHOH, R₄ is H and R₃ is C₅ alkylthen Y is not CH₂ (reduced paradol); (5) R₁ is OCH₃, R₂ is OH, W=CH₂CH₂,X is C=O, R₄ is H then Y is not C(OH)CH₃; (6) R₁ is OC₁₋₄ alkyl, R₂ isOH or OAcyl, W=CH═CH and X=C═O, R₃ is C₂₋₉ alkyl, R₄ is H, then Y is notCHOH; (7) R₁=R₂ is OH, W=CH═CH and X=C═O, R₃ is C₉ alkyl, R₄ is H, thenY is not CHOH ([10]-nordehydrogingerols); (8) R₁=R₂ is OH, W=CH₂CH₂ andX=C═O, R₃ is C₂₋₁₂ alkyl, R₄ is H, then Y is not CHOH (norgingerols);and (9) R₁ is OC₁₋₄ alkyl or OH, R₂ is OH, W is CH₂CH₂, R₃ is C₂₋₁₂alkyl, R₄ is H and X is CHOH, then Y is not CHOH (gingerdiols ornorgingerdiols).
 2. A method for inhibition of platelet aggregation in asubject in need of such inhibition comprising administering to saidsubject an amount effective to inhibit platelet aggregation of acompound of formula (I) or a pharmaceutically acceptable derivativethereof:

where R₁ is H, OH, OC₁₋₄alkyl, NO₂ R₂ is OH, OC₂₋₄alkyl, OC═OC₁₋₄alkylor OC═OPh where the Ph can be optionally substituted by halogen, C₁₋₃alkyl or NO₂; R₁ and R₂ along with the two carbon atoms of the phenylring to which they are attached can combine to form a 5 or 6 memberedheterocyclic ring comprising 1 or 2 heteroatoms selected from O, S or N;R₃ is C₂₋₁₂alkyl, C₂₋₁₂alkenyl or C₂₋₁₂alkynyl each optionallysubstituted by one or more substituents selected from —OR, ═O, nitro,halogen, —NRR′, —COOR or —CONRR′, where R and R′ are H or C₁₋₄alkyl; R₃may be a linking group of a bis compound where R₃ is C₂₋₁₂alkylene,C₂₋₁₂alkenylene or C₂₋₁₂alkynylene each optionally substituted by one ormore substituents selected from —OR, ═O, nitro, halogen, —NRR′, —COOR or—CONRR′ where R and R′ are H or C₁₋₄alkyl; R₄ is H, CH₃, OH or ═O; whenR₄ is ═O, then the carbon to which R₄ is attached is not bonded to H; Wis C(═O)—CH₂, CH═CH—, CH₂CO, CH(OH)—CH₂, C(CH₃)(OH)CH₂, CH₂CH(OH),CH₂C(CH₃)OH, CO, CHOH C(CH₃)(OH), CH₂, CH₂CH₂; X is —CH—OH, C(CH₃)OH,CH₂, CH(CH₃) or —C═O; Y is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;provided that one of W, X or Y has an OH group.
 3. A pharmaceuticalcomposition comprising a compound of claim 1 or a pharmaceuticallyacceptable derivative thereof and a pharmaceutically acceptable carrier.4. A compound selected from the group consisting of:1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol;1-(4-hydroxy-3-methoxyphenyl)dodecan-5-ol;3-methyl-1-(4-hydroxy-3-methoxyphenyl)undecan-3-ol;3-methyl-1-(4-hydroxy-3-methoxyphenyl)tridecan-3-ol;3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-5-one;3-hydrox-y-1-(4-hydroxy-3-methoxyphenyl)decan-1-one;3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-1-one;1-hydroxy-1-(4-hydroxy-3-methox-yphenyl)undecan-2-one;2-hydroxy-1-(4-hydroxy-3-methoxyphenyl)undecan-1-one;5-hydroxy-1-(4-hydroxyphenyl)decan-3-one;5-hydroxy-1-(4-hydroxyphenyl)dodecan-3-one;5-hydroxy-1-(4-hydroxyphenyl)dodecan-1-ene-3-one;5-hydroxy-1-(3,4-methylenedioxyphenyl)dodecan-3-one;5,12-dihydroxy-1,16-bis(4-hydroxy-3-methoxyphenyl)hexadecane-3,14-dione;1-(4-hydroxy-3-methoxyphenyl)dodecane-1,4-diene-3-one;2-hydrox-y-1-(3,4-dimethoxy-phenyl)dodecan-3-one;2-hydroxy-1-(3,4-dimethoxyphenyl)undecan-4-one; and1-(3,4-dimethoxyphenyl)dodecan-2-ol.
 5. A compound of claim 4 selectedfrom the group consisting of: 1-(4-hydroxy-3-methoxyphenyl)dodecan-3-ol;3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-5-one;3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-1-one; and3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-1-one.
 6. A method fortreatment or prophylaxis of pain by action on sensory nerves or throughanti-inflammatory action or through neurokinin inhibitory action in asubject in need of such treatment or prophylaxis comprisingadministering to said subject an effective amount of a compound offormula (I) or a pharmaceutically acceptable derivative thereof:

where R₁ is H, OH, OC₁₋₄alkyl, NO₂, R₂ is OH, OC₁₋₄alkyl, OC═OC₁₋₄alkylor OC═OPh where the Ph can be optionally substituted by halogen, C₁₋₂alkyl or NO₂; R₁ and R₂ along with the two carbon atoms of the phenylring to which they are attached can combine to form a 5 or 6 memberedheterocyclic ring comprising 1 or 2 heteroatoms selected from O, S or N;R₃ is C₂₋₁₂alkyl, C₂₋₁₂alkenyl or C₂₋₁₂alkynyl each optionallysubstituted by one or more substituents selected from —OR, ═O, nitro,halogen, —NRR′, —COOR or —CONRR′ where R and R′ are H or C₁₋₄alkyl; R₃may be a linking group of a bis compound where R₃ is C₂₋₁₂alkylene,C₂₋₁₂alkenylene or C₂₋₁₂alkynylene each optionally substituted by one ormore substituents selected from —OR, ═O, nitro, halogen, —NRR′, —COOR or—CONRR′ where R and R′ are H or C₁₋₄alkyl; R₄ is H, CH₃, OH or ═O; whenR₄ is ═O, then the carbon to which R₄ is attached is not bonded to H; Wis C(═O)—CH₂, CH═CH—, CH₂O, CH(OH)—CH₂, C(CH₃)(OH)CH₂, CH₂CH(OH),CH₂C(CH₃)OH, CO, CHOH, C(CH₃)(OH), CH₂, CH₂CH₂; X is —CH—OH, C(CH₃)OH,CH₂, CH(CH₃) or —C═O; Y is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;provided that one of W, X or Y has an OH group.
 7. A method according toclaim 6 wherein said compound or pharmaceutically acceptable derivativethereof is used as an analgesic.
 8. A method for treatment orprophylaxis of cardiovascular disease in a subject in need of suchtreatment or prophylaxis comprising administering to said subject aneffective amount of a compound of formula (I) or a pharmaceuticallyacceptable derivative thereof:

where R₁ is H, OH, OC₁₋₄alkyl, NO₂ R₂ is OH, OC₁₋₄alkyl, OC═OC₁₋₄alkylor OC═OPh where the Ph can be optionally substituted by halogen, C₁₋₃alkyl or NO₂; R₁ and R₂ along with the two carbon atoms of the phenylring to which they are attached can combine to form a 5 or 6 memberedheterocyclic ring comprising 1 or 2 heteroatoms selected from O, S or N;R₃ is C₂₋₁₂alkyl, C₂₋₁₂alkenyl or C₂₋₁₂alkynyl each optionallysubstituted by one or more substituents selected from —OR, ═O, nitro,halogen, —NRR′, —COOR or —CONRR′ where R and R′ are H or C₁₋₄alkyl; R₃may be a linking group of a bis compound where R₃ is C₂₋₁₂alkylene,C₂₋₁₂alkenylene or C₂₋₁₂alkynylene each optionally substituted by one ormore substituents selected from —OR, ═O, nitro, halogen, —NRR′, —COOR or—CONRR′ where R and R′ are H or C₂₋₄alkyl; R₄ is H, CH₃, OH or =O; whenR₄ is ═O, then the carbon to which R₄ is attached is not bonded to H; Wis C(═O)—CH₂, CH═CH—, CH₂CO, CH(OH)—CH₂, C(CH₃)(OH)CH₂, CH₂CH(OH),CH₂C(CH₃)OH, CO, CHOH, C(CH₃)(OH), CH₂, CH₂CH₂; X is —CH—OH, C(CH₃)OH,CH₂, CH(CH₃) or —C═O; Y is —CH—OH, C(CH₃)OH, CH₂, CH(CH₃) or —C═O;provided that one of W, X or Y has an OH group.
 9. A process ofpreparing a compound having a formula selected from the following group:

n=1-10 which method comprises treating ginger extract with heat or acid,followed by treating the extract with a microorganism or a microbialenzyme.